Isolated human Ras-like proteins, nucleic acid molecules encoding these human Ras-like proteins, and uses thereof

ABSTRACT

The present invention provides amino acid sequences of polypeptides that are encoded by genes within the human genome, the Ras-like protein polypeptides of the present invention. The present invention specifically provides isolated polypeptide and nucleic acid molecules, methods of identifying orthologs and paralogs of the Ras-like protein polypeptides, and methods of identifying modulators of the Ras-like protein polypeptides.

FIELD OF THE INVENTION

[0001] The present invention is in the field of Ras-like proteins thatare related to the guanine nucleotide exchange factor (GEF) subfamily,recombinant DNA molecules and protein production. The present inventionspecifically provides novel Ras-like protein polypeptides and proteinsand nucleic acid molecules encoding such peptide and protein molecules,all of which are useful in the development of human therapeutics anddiagnostic compositions and methods.

BACKGROUND OF THE INVENTION

[0002] Ras-like proteins, particularly members of the GEF subfamilies,are a major target for drug action and development. Accordingly, it isvaluable to the field of pharmaceutical development to identify andcharacterize previously unknown members of these subfamily of Ras-likeproteins. The present invention advances the state of the art byproviding a previously unidentified human Ras-like proteins that havehomology to members of the GEF subfamilies.

Ras Protein

[0003] Ras proteins are small regulatory GTP-binding proteins, or smallG proteins, which belong to the Ras protein superfamily. They aremonomeric GTPases, but their GTPase activity is very slow (less than oneGTP molecule per minute).

[0004] Ras proteins are key relays in the signal transducing cascadeinduced by the binding of a ligand to specific receptors such asreceptor tyrosine kinases (RTKs), since they trigger the MAP kinasecascade. The ligand can be a growth factor (epidermal growth factor(EGF), platelet-derived growth factor (PDGF) . . . ), insulin, aninterleukin (IL), granulocyte colony-stimulating factor (G-CSF),granulocyte/macrophage colony-stimulating factor (GM-CSF). . .

[0005] Ras proteins contain sequences highly conserved during evolution.Their tertiary structure includes ten loops connecting six strands ofbeta-sheet and five alpha helices.

[0006] In mammalians, there are four Ras proteins, which are encoded byHa-ras, N-ras, Ki-rasA and Ki-rasB genes. They are composed of about 170residues and have a relative molecular mass of 21 kD. Ras proteinscontain covalently attached modified lipids allowing these proteins tobind to the plasma membrane. Ha-Ras has a C-terminal farnesyl group, aC-terminal palmitoyl group and a N-terminal myristoyl group. InKi-Ras(B), a C-terminal polylysine domain replaces the palmitoyl group.

[0007] Ras proteins alternate between an inactive form bound to GDP andan active form bound to GTP. Their activation results from reactionsinduced by a guanine nucleotide-exchange factor (GEF). Theirinactivation results from reactions catalyzed by a GTPase-activatingprotein (GAP).

[0008] When a Ras protein is activated by a GEF such as a Sos protein,the N-terminal region of a serine/threonine kinase, called “Rafprotein”, can bind to Ras protein. The C-terminal region of theactivated Raf thus formed binds to another protein, MEK, andphosphorylates it on both specific tyrosine and serine residues. ActiveMEK phosphorylates and activates, in turn, a MAP kinase (ERK1 or ERK2),which is also a serine/threonine kinase. This phosphorylation occurs onboth specific tyrosine and threonine residues of MAP kinase.

[0009] MAP kinase phosphorylates many different proteins, especiallynuclear transcription factors (TFs) which regulate expression of manygenes during cell proliferation and differentiation.

[0010] Recent researches suggest that, in mammalians, phosphatidylinositol 3′-kinase (PI3-kinase) might be a target of Ras protein,instead of Raf protein. In certain mutations, the translation of rasgenes may produce oncogenic Ras proteins.

Ras-like Protein

[0011] Guanine nucleotide-binding proteins (GTP-binding proteins, or Gproteins) participate in a wide range of regulatory functions includingmetabolism, growth, differentiation, signal transduction, cytoskeletalorganization, and intracellular vesicle transport and secretion. Theseproteins control diverse sets of regulatory pathways in response tohormones, growth factors, neuromodulators, or other signaling molecules.When these molecules bind to transmembrane receptors, signals arepropagated to effector molecules by intracellular signal transducingproteins. Many of these signal transducing proteins are members of theRas superfamily.

[0012] The Ras superfamily is a class of low molecular weight (LMW)GTP-binding proteins which consist of 21-30 kDa polypeptides. Theseproteins regulate cell growth, cell cycle control, protein secretion,and intracellular vesicle interaction. In particular, the LMWGTP-binding proteins activate cellular proteins by transducing mitogenicsignals involved in various cell functions in response to extracellularsignals from receptors (Tavitian, A. (1995) C. R. Seances Soc. Biol.Fil. 189:7-12). During this process, the hydrolysis of GTP acts as anenergy source as well as an on-off switch for the GTPase activity of theLMW GTP-binding proteins.

[0013] The Ras superfamily is comprised of five subfamilies: Ras, Rho,Ran, Rab, and ADP-ribosylation factor (ARF). Specifically, Ras genes areessential in the control of cell proliferation. Mutations in Ras geneshave been associated with cancer. Rho proteins control signaltransduction in the process of linking receptors of growth factors toactin polymerization which is necessary for cell division. Rab proteinscontrol the translocation of vesicles to and from membranes for proteinlocalization, protein processing, and secretion. Ran proteins arelocalized to the cell nucleus and play a key role in nuclear proteinimport, control of DNA synthesis, and cell-cycle progression. ARF andARF-like proteins participate in a wide variety of cellular functionsincluding vesicle trafficking, exocrine secretion, regulation ofphospholipase activity, and endocytosis.

[0014] Despite their sequence variations, all five subfamilies of theRas superfamily share conserved structural features. Four conservedsequence regions (motifs I-IV) have been studied in the LMW GTP-bindingproteins. Motif I is the most variable but has the conserved sequence,GXXXXGK. The lysine residue is essential in interacting with the .beta.-and .gamma.-phosphates of GTP. Motif II, III, and IV contain highlyconserved sequences of DTAGQ, NKXD, and EXSAX, respectively.Specifically, Motif II regulates the binding of gamma-phosphate of GTP;Motif III regulates the binding of GTP; and Motif IV regulates theguanine base of GTP. Most of the membrane-bound LMW GTP-binding proteinsgenerally require a carboxy terminal isoprenyl group for membraneassociation and biological activity. The isoprenyl group is addedposttranslationally through recognition of a terminal cysteine residuealone or a terminal cysteine-aliphatic amino acid-aliphatic aminoacid-any amino acid (CAAX) motif. Additional membrane-binding energy isoften provided by either internal palmitoylation or a carboxy terminalcluster of basic amino acids. The LMW GTP-binding proteins also have avariable effector region, located between motifs I and II, which ischaracterized as the interaction site for guanine nucleotide exchangefactors (GEFs) or GTPase-activating proteins (GAPs). GEFs induce therelease of GDP from the active form of the G protein, whereas GAPsinteract with the inactive form by stimulating the GTPase activity ofthe G protein.

[0015] The ARF subfamily has at least 15 distinct members encompassingboth ARF and ARF-like proteins. ARF proteins identified to date exhibithigh structural similarity and ADP-ribosylation enhancing activity. Incontrast, several ARF-like proteins lack ADP-ribosylation enhancingactivity and bind GTP differently. An example of ARF-like proteins is arat protein, ARL184. ARL184 has been shown to have a molecular weight of22 kDa and four functional GTP-binding sites (Icard-Liepkalns, C. et al.(1997) Eur. J. Biochem. 246:388-393). ARL184 is active in both thecytosol and the Golgi apparatus and is closely associated withacetylcholine release, suggesting that ARL184 is a potential regulatoryprotein associated with Ca.sup.2+-dependent release of acetylcholine.

[0016] A number of Rho GTP-binding proteins have been identified inplasma membrane and cytoplasm. These include RhoA, B and C, and D, rhoG,rac 1 and 2, G25K-A and B, and TC10 (Hall, A. et al. (1993) Philos.Trans. R. Soc. Lond. (Biol.) 340:267-271). All Rho proteins have a CAAXmotif which binds a prenyl group and either a palmitoylation site or abasic amino acid-rich region, suggesting their role inmembrane-associated functions. In particular, RhoD is a protein whichfunctions in early endosome motility and distribution by inducingrearrangement of actin cytoskeleton and cell surface (Murphy, C. et al.(1996) Nature 384:427-432). During cell adhesion, the Rho proteins areessential for triggering focal complex assembly and integrin-dependentsignal transduction (Hotchin, N. A. and Hall, A. (1995) J. Cell Biol.131:1857-1865).

[0017] The Ras subfamily proteins already indicated supra are essentialin transducing signals from receptor tyrosine kinases (RTKs) to a seriesof serine/threonine kinases which control cell growth anddifferentiation. Mutant Ras proteins, which bind but cannot hydrolyzeGTP, are permanently activated and cause continuous cell proliferationor cancer. TC21, a Ras-like protein, is found to be highly expressed ina human teratocarcinoma cell line (Drivas, G. T. et al. (1990) Mol.Cell. Biol. 10:1793-1798). Rin and Rit are characterized asmembrane-binding, Ras-like proteins without the lipid-binding CAAX motifand carboxy terminal cysteine (Lee, C. -H. J. et al. (1996) J. Neurosci.16:6784-6794). Further, Rin is shown to localize in neurons and havecalcium-dependant calmodulin-binding activity.

Guanine Nucleotide Exchange Factor (GEF) Proteins

[0018] The novel human protein, and encoding gene, provided by thepresent invention is a novel alternative splice form of a known cDNA(Genbank gi:7209309; see the alignment in FIG. 2). This protein shares ahigh degree of similarity to the RhoGEF proteins, particularly the FGD1subfamily. FGD1 is mutated in faciogenital dysplasia (also known asAarskog syndrome), which is an X-linked developmental disorder that ischaracterized by defects in skeletal structure formation. Guaninenucleotide exchange factors (GEF) modulate the activity of Ras proteins.The FGD1 gene encodes a GEF that activates the Rho GTPase Cdc42. RhoGTPases are members of the Ras family and are believed to be involved incytoskeletal restructuring. Specifically, FGD1, via Cdc42, modulatesactin cytoskeleton and stimulates the c-Jun N-terminal kinase signalingcascade, thereby modulating cell growth and differentiation. Proteins ofthe FGD1 family contain adjacent RhoGEF and pleckstrin homology domains,a second carboxy-terminal pleckstrin homology domain, and achracteristic FYVE domain; these domains are thought to form a canonicalcore structure. Fgd3, a member of the FGD1 family, stimulates conversionof fibroblasts to filopodia, which are actin microspikes formed uponstimulation of Cdc42. It has been suggested that FGD1 proteins may playimportant roles in embryonic development (Pasteris et al., Gene 242(1-2), 237-247 (2000) and Pasteris et al., Genomics 60:57-66, 1999).

[0019] The discovery of new human Ras-like proteins and thepolynucleotides which encode them satisfies a need in the art byproviding new compositions which are useful in the diagnosis,prevention, and treatment of inflammation and disorders associated withcell proliferation and apoptosis.

SUMMARY OF THE INVENTION

[0020] The present invention is based in part on the identification of anovel alternative splice form of human Ras-like protein, polypeptidesand proteins that are related to the guanine nucleotide exchange factor(GEF) protein subfamily, as well as allelic variants and other mammalianorthologs thereof. The unique peptide sequences encoding the novelalternative splice form provided herein, and nucleic acid sequences thatencode these peptides, can be used as models for the development ofhuman therapeutic targets, aid in the identification of therapeuticproteins, and serve as targets for the development of human therapeuticagents that modulate Ras-like protein activity in cells and tissues thatexpress the Ras-like protein. Experimental data as provided in FIG. 1indicates expression in humans in B-cells from tonsils, adrenal cortexcarcinomas, Jurkat T-cells, prostate adenocarcinomas, lung (includingnormal and small cell carcinoma), marrow, placenta, and leukocytes.

DESCRIPTION OF THE FIGURE SHEETS

[0021]FIG. 1 provides the nucleotide sequence of a cDNA molecule thatencodes the Ras-like protein of the present invention. (SEQ ID NO:1) Inaddition, structure and functional information is provided, such as ATGstart, stop and tissue distribution, where available, that allows one toreadily determine specific uses of inventions based on this molecularsequence. Experimental data as provided in FIG. 1 indicates expressionin humans in B-cells from tonsils, adrenal cortex carcinomas, JurkatT-cells, prostate adenocarcinomas, lung (including normal and small cellcarcinoma), marrow, placenta, and leukocytes.

[0022]FIG. 2 provides the predicted amino acid sequence of the Ras-likeprotein of the present invention. (SEQ ID NO:2) In addition structureand functional information such as protein family, function, andmodification sites is provided where available, allowing one to readilydetermine specific uses of inventions based on this molecular sequence.

[0023]FIG. 3 provides genomic sequences that span the gene encoding theRas-like protein of the present invention. (SEQ ID NO:3) In additionstructure and functional information, such as intron/exon structure,promoter location, etc., is provided where available, allowing one toreadily determine specific uses of inventions based on this molecularsequence.

DETAILED DESCRIPTION OF THE INVENTION General Description

[0024] The present invention is based on the sequencing of the humangenome. During the sequencing and assembly of the human genome, analysisof the sequence information revealed previously unidentified fragmentsof the human genome that encode peptides that share structural and/orsequence homology to protein/peptide/domains identified andcharacterized within the art as being a Ras-like protein or part of aRas-like protein and are related to the guanine nucleotide exchangefactor (GEF) subfamily. Utilizing these sequences, additional genomicsequences were assembled and transcript and/or cDNA sequences wereisolated and characterized. Based on this analysis, the presentinvention provides amino acid sequences of human Ras-like proteinpolypeptides that represent a novel alternative splice form of a GEFprotein, nucleic acid sequences in the form of transcript sequences,cDNA sequences and/or genomic sequences that encode the novelalternative splice form, nucleic acid variation (allelic information),tissue distribution of expression, and information about the closest artknown protein/peptide/domain that has structural or sequence homology tothe Ras-like protein of the present invention.

[0025] In addition to being previously unknown, the peptides that areprovided in the present invention are selected based on their ability tobe used for the development of commercially important products andservices. Specifically, the present peptides are selected based onhomology and/or structural relatedness to known Ras-like proteins of theGEF subfamily and the expression pattern observed. Experimental data asprovided in FIG. 1 indicates expression in humans in B-cells fromtonsils, adrenal cortex carcinomas, Jurkat T-cells, prostateadenocarcinomas, lung (including normal and small cell carcinoma),marrow, placenta, and leukocytes. The art has clearly established thecommercial importance of members of this family of proteins and proteinsthat have expression patterns similar to that of the present gene. Someof the more specific features of the peptides of the present invention,and the uses thereof, are described herein, particularly in theBackground of the Invention and in the annotation provided in theFigures, and/or are known within the art for each of the known GEFfamily or subfamily of Ras-like proteins.

SPECIFIED EMBODIMENTS Peptide Molecules

[0026] The present invention provides nucleic acid sequences that encodeprotein molecules that have been identified as being members of theRas-like protein family and are related to the GEF subfamily (proteinsequences are provided in FIG. 2, transcript/cDNA sequences are providedin FIG. 1 and genomic sequences are provided in FIG. 3). The peptidesequences provided in FIG. 2, as well as the obvious variants describedherein, particularly allelic variants as identified herein and using theinformation in FIG. 3, will be referred herein as the Ras-like proteinsor peptides of the present invention, Ras-like proteins or peptides, orpeptides/proteins of the present invention.

[0027] The present invention provides isolated peptide and proteinmolecules that consist of, consist essentially of, or comprise the aminoacid sequences of the Ras-like protein polypeptide disclosed in the FIG.2, (encoded by the nucleic acid molecule shown in FIG. 1,transcript/cDNA or FIG. 3, genomic sequence), as well as all obviousvariants of these peptides that are within the art to make and use. Someof these variants are described in detail below.

[0028] As used herein, a peptide is said to be “isolated” or “purified”when it is substantially free of cellular material or free of chemicalprecursors or other chemicals. The peptides of the present invention canbe purified to homogeneity or other degrees of purity. The level ofpurification will be based on the intended use. The critical feature isthat the preparation allows for the desired function of the peptide,even if in the presence of considerable amounts of other components.

[0029] In some uses, “substantially free of cellular material” includespreparations of the peptide having less than about 30% (by dry weight)other proteins (i.e., contaminating protein), less than about 20% otherproteins, less than about 10% other proteins, or less than about 5%other proteins. When the peptide is recombinantly produced, it can alsobe substantially free of culture medium, i.e., culture medium representsless than about 20% of the volume of the protein preparation.

[0030] The language “substantially free of chemical precursors or otherchemicals” includes preparations of the peptide in which it is separatedfrom chemical precursors or other chemicals that are involved in itssynthesis. In one embodiment, the language “substantially free ofchemical precursors or other chemicals” includes preparations of theRas-like protein polypeptide having less than about 30% (by dry weight)chemical precursors or other chemicals, less than about 20% chemicalprecursors or other chemicals, less than about 10% chemical precursorsor other chemicals, or less than about 5% chemical precursors or otherchemicals.

[0031] The isolated Ras-like protein polypeptide can be purified fromcells that naturally express it, purified from cells that have beenaltered to express it (recombinant), or synthesized using known proteinsynthesis methods. Experimental data as provided in FIG. 1 indicatesexpression in humans in B-cells from tonsils, adrenal cortex carcinomas,Jurkat T-cells, prostate adenocarcinomas, lung (including normal andsmall cell carcinoma), marrow, placenta, and leukocytes. For example, anucleic acid molecule encoding the Ras-like protein polypeptide iscloned into an expression vector, the expression vector introduced intoa host cell and the protein expressed in the host cell. The protein canthen be isolated from the cells by an appropriate purification schemeusing standard protein purification techniques. Many of these techniquesare described in detail below.

[0032] Accordingly, the present invention provides proteins that consistof the amino acid sequences provided in FIG. 2 (SEQ ID NO:2), forexample, proteins encoded by the transcript/cDNA nucleic acid sequencesshown in FIG. 1 (SEQ ID NO:1) and the genomic sequences provided in FIG.3 (SEQ ID NO:3). The amino acid sequence of such a protein is providedin FIG. 2. A protein consists of an amino acid sequence when the aminoacid sequence is the final amino acid sequence of the protein.

[0033] The present invention further provides proteins that consistessentially of the amino acid sequences provided in FIG. 2 (SEQ IDNO:2), for example, proteins encoded by the transcript/cDNA nucleic acidsequences shown in FIG. 1 (SEQ ID NO:1) and the genomic sequencesprovided in FIG. 3 (SEQ ID NO:3). A protein consists essentially of anamino acid sequence when such an amino acid sequence is present withonly a few additional amino acid residues, for example from about 1 toabout 100 or so additional residues, typically from 1 to about 20additional residues in the final protein.

[0034] The present invention further provides proteins that comprise theamino acid sequences provided in FIG. 2 (SEQ ID NO:2), for example,proteins encoded by the transcript/cDNA nucleic acid sequences shown inFIG. 1 (SEQ ID NO:1) and the genomic sequences provided in FIG. 3 (SEQID NO:3). A protein comprises an amino acid sequence when the amino acidsequence is at least part of the final amino acid sequence of theprotein. In such a fashion, the protein can be only the peptide or haveadditional amino acid molecules, such as amino acid residues (contiguousencoded sequence) that are naturally associated with it or heterologousamino acid residues/peptide sequences. Such a protein can have a fewadditional amino acid residues or can comprise several hundred or moreadditional amino acids. The preferred classes of proteins that arecomprised of the Ras-like protein polypeptide of the present inventionare the naturally occurring mature proteins. A brief description of howvarious types of these proteins can be made/isolated is provided below.

[0035] The Ras-like protein polypeptides of the present invention can beattached to heterologous sequences to form chimeric or fusion proteins.Such chimeric and fusion proteins comprise a Ras-like proteinpolypeptide operatively linked to a heterologous protein having an aminoacid sequence not substantially homologous to the Ras-like proteinpolypeptide. “Operatively linked” indicates that the Ras-like proteinpolypeptide and the heterologous protein are fused in-frame. Theheterologous protein can be fused to the N-terminus or C-terminus of theRas-like protein polypeptide.

[0036] In some uses, the fusion protein does not affect the activity ofthe Ras-like protein polypeptide per se. For example, the fusion proteincan include, but is not limited to, enzymatic fusion proteins, forexample beta-galactosidase fusions, yeast two-hybrid GAL fusions,poly-His fusions, MYC-tagged, HI-tagged and Ig fusions. Such fusionproteins, particularly poly-His fusions, can facilitate the purificationof recombinant Ras-like protein polypeptide. In certain host cells(e.g., mammalian host cells), expression and/or secretion of a proteincan be increased by using a heterologous signal sequence.

[0037] A chimeric or fusion protein can be produced by standardrecombinant DNA techniques. For example, DNA fragments coding for thedifferent protein sequences are ligated together in-frame in accordancewith conventional techniques. In another embodiment, the fusion gene canbe synthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers which give rise to complementaryoverhangs between two consecutive gene fragments which can subsequentlybe annealed and re-amplified to generate a chimeric gene sequence (seeAusubel et al., Current Protocols in Molecular Biology, 1992). Moreover,many expression vectors are commercially available that already encode afusion moiety (e.g., a GST protein). A Ras-like proteinpolypeptide-encoding nucleic acid can be cloned into such an expressionvector such that the fusion moiety is linked in-frame to the Ras-likeprotein polypeptide.

[0038] As mentioned above, the present invention also provides andenables obvious variants of the amino acid sequence of the peptides ofthe present invention, such as naturally occurring mature forms of thepeptide, allelic/sequence variants of the peptides, non-naturallyoccurring recombinantly derived variants of the peptides, and orthologsand paralogs of the peptides. Such variants can readily be generatedusing art know techniques in the fields of recombinant nucleic acidtechnology and protein biochemistry. It is understood, however, thatvariants exclude any amino acid sequences disclosed prior to theinvention.

[0039] Such variants can readily be identified/made using moleculartechniques and the sequence information disclosed herein. Further, suchvariants can readily be distinguished from other peptides based onsequence and/or structural homology to the Ras-like protein polypeptidesof the present invention. The degree of homology/identity present willbe based primarily on whether the peptide is a functional variant ornon-functional variant, the amount of divergence present in the paralogfamily, and the evolutionary distance between the orthologs.

[0040] To determine the percent identity of two amino acid sequences ortwo nucleic acid sequences, the sequences are aligned for optimalcomparison purposes (e.g., gaps can be introduced in one or both of afirst and a second amino acid or nucleic acid sequence for optimalalignment and non-homologous sequences can be disregarded for comparisonpurposes). In a preferred embodiment, the length of a reference sequencealigned for comparison purposes is at least 30%, 40%, 50%, 60%, 70%,80%, or 90% or more of the length of the reference sequence. The aminoacid residues or nucleotides at corresponding amino acid positions ornucleotide positions are then compared. When a position in the firstsequence is occupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein amino acid or nucleic acid“identity” is equivalent to amino acid or nucleic acid “homology”). Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences.

[0041] The comparison of sequences and determination of percent identityand similarity between two sequences can be accomplished using amathematical algorithm. (Computational Molecular Biology, Lesk, A. M.,ed., Oxford University Press, New York, 1988; Biocomputing: Informaticsand Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of sequence Data, Part 1, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991). In a preferred embodiment, the percent identity betweentwo amino acid sequences is determined using the Needleman and Wunsch (JMol Biol. (48):444-453 (1970)) algorithm which has been incorporatedinto the GAP program in the GCG software package (available athttp://www.gcg.com), using either a Blossom 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, thepercent identity between two nucleotide sequences is determined usingthe GAP program in the GCG software package (Devereux, J., et al.,Nucleic Acids Res. 12(1):387 (1984)) (available at http://www.gcg.com),using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, thepercent identity between two amino acid or nucleotide sequences isdetermined using the algorithm of E. Meyers and W. Miller (CABIOS,4:11-17 (1989)) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4.

[0042] The nucleic acid and protein sequences of the present inventioncan further be used as a “query sequence” to perform a search againstsequence databases to, for example, identify other family members orrelated sequences. Such searches can be performed using the NBLAST andXBLAST programs (version 2.0) of Altschul, et al. (J. Mol. Biol.215:403-10 (1990)). BLAST nucleotide searches can be performed with theNBLAST program, score=100, word length=12 to obtain nucleotide sequenceshomologous to the nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the XBLAST program, score=50, wordlength=3, to obtain amino acid sequences homologous to the proteins ofthe invention. To obtain gapped alignments for comparison purposes,Gapped BLAST can be utilized as described in Altschul et al. (NucleicAcids Res. 25(17):3389-3402 (1997)). When utilizing BLAST and gappedBLAST programs, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.

[0043] Full-length pre-processed forms, as well as mature processedforms, of proteins that comprise one of the peptides of the presentinvention can readily be identified as having complete sequence identityto one of the Ras-like protein polypeptides of the present invention aswell as being encoded by the same genetic locus as the Ras-like proteinpolypeptide provided herein. The gene encoding the novel Ras-likeprotein of the present invention is located on a genome component thathas been mapped to human chromosome 9 (as indicated in FIG. 3), which issupported by multiple lines of evidence, such as STS and BAC map data.

[0044] Allelic variants of a Ras-like protein polypeptide can readily beidentified as being a human protein having a high degree (significant)of sequence homology/identity to at least a portion of the Ras-likeprotein polypeptide as well as being encoded by the same genetic locusas the Ras-like protein polypeptide provided herein. Genetic locus canreadily be determined based on the genomic information provided in FIG.3, such as the genomic sequence mapped to the reference human. The geneencoding the novel Ras-like protein of the present invention is locatedon a genome component that has been mapped to human chromosome 9 (asindicated in FIG. 3), which is supported by multiple lines of evidence,such as STS and BAC map data. As used herein, two proteins (or a regionof the proteins) have significant homology when the amino acid sequencesare typically at least about 70-80%, 80-90%, and more typically at leastabout 90-95% or more homologous. A significantly homologous amino acidsequence, according to the present invention, will be encoded by anucleic acid sequence that will hybridize to a Ras-like proteinpolypeptide encoding nucleic acid molecule under stringent conditions asmore fully described below.

[0045] Paralogs of a Ras-like protein polypeptide can readily beidentified as having some degree of significant sequencehomology/identity to at least a portion of the Ras-like proteinpolypeptide, as being encoded by a gene from humans, and as havingsimilar activity or function. Two proteins will typically be consideredparalogs when the amino acid sequences are typically at least about40-50%, 50-60%, and more typically at least about 60-70% or morehomologous through a given region or domain. Such paralogs will beencoded by a nucleic acid sequence that will hybridize to a Ras-likeprotein polypeptide encoding nucleic acid molecule under moderate tostringent conditions as more fully described below.

[0046] Orthologs of a Ras-like protein polypeptide can readily beidentified as having some degree of significant sequencehomology/identity to at least a portion of the Ras-like proteinpolypeptide as well as being encoded by a gene from another organism.Preferred orthologs will be isolated from mammals, preferably primates,for the development of human therapeutic targets and agents. Suchorthologs will be encoded by a nucleic acid sequence that will hybridizeto a Ras-like protein polypeptide encoding nucleic acid molecule undermoderate to stringent conditions, as more fully described below,depending on the degree of relatedness of the two organisms yielding theproteins.

[0047] Non-naturally occurring variants of the Ras-like proteinpolypeptides of the present invention can readily be generated usingrecombinant techniques. Such variants include, but are not limited todeletions, additions and substitutions in the amino acid sequence of theRas-like protein polypeptide. For example, one class of substitutions isconserved amino acid substitutions. Such substitutions are those thatsubstitute a given amino acid in a Ras-like protein polypeptide byanother amino acid of like characteristics. Typically seen asconservative substitutions are the replacements, one for another, amongthe aliphatic amino acids Ala, Val, Leu, and Ile; interchange of thehydroxyl residues Ser and Thr, exchange of the acidic residues Asp andGlu, substitution between the amide residues Asn and Gln, exchange ofthe basic residues Lys and Arg, replacements among the aromatic residuesPhe, Tyr, and the like. Guidance concerning which amino acid changes arelikely to be phenotypically silent are found in Bowie et al., Science247:1306-1310 (1990).

[0048] Variant Ras-like protein polypeptides can be fully functional orcan lack function in one or more activities. Fully functional variantstypically contain only conservative variations or variations innon-critical residues or in non-critical regions. Functional variantscan also contain substitution of similar amino acids that result in nochange or an insignificant change in function. Alternatively, suchsubstitutions may positively or negatively affect function to somedegree.

[0049] Non-functional variants typically contain one or morenon-conservative amino acid substitutions, deletions, insertions,inversions, or truncation or a substitution, insertion, inversion, ordeletion in a critical residue or critical region.

[0050] Amino acids that are essential for function can be identified bymethods known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham et al., Science 244:1081-1085(1989)). The latter procedure introduces single alanine mutations atevery residue in the molecule. The resulting mutant molecules are thentested for biological activity such as receptor binding or in vitroproliferative activity. Sites that are critical for ligand-receptorbinding can also be determined by structural analysis such ascrystallography, nuclear magnetic resonance, or photoaffinity labeling(Smith et aL, J. Mol. Biol. 224:899-904 (1992); de Vos et aL. Science255:306-312 (1992)).

[0051] The present invention further provides fragments of the Ras-likeprotein polypeptides, in addition to proteins and peptides that compriseand consist of such fragments. Particularly those comprising theresidues identified in FIG. 2. The fragments to which the inventionpertains, however, are not to be construed as encompassing fragmentsthat have been disclosed publicly prior to the present invention.

[0052] As used herein, a fragment comprises at least 8, 10, 12, 14, 16or more contiguous amino acid residues from a Ras-like proteinpolypeptide. Such fragments can be chosen based on the ability to retainone or more of the biological activities of the Ras-like proteinpolypeptide, or can be chosen for the ability to perform a function,e.g., act as an immunogen. Particularly important fragments arebiologically active fragments, peptides that are, for example about 8 ormore amino acids in length. Such fragments will typically comprise adomain or motif of the Ras-like protein polypeptide, e.g., active site.Further, possible fragments include, but are not limited to, domain ormotif containing fragments, soluble peptide fragments, and fragmentscontaining immunogenic structures. Predicted domains and functionalsites are readily identifiable by computer programs well known andreadily available to those of skill in the art (e.g., PROSITE, HMMer,eMOTIF, etc.). The results of one such analysis are provided in FIG. 2.

[0053] Polypeptides often contain amino acids other than the 20 aminoacids commonly referred to as the 20 naturally occurring amino acids.Further, many amino acids, including the terminal amino acids, may bemodified by natural processes, such as processing and otherpost-translational modifications, or by chemical modification techniqueswell known in the art. Common modifications that occur naturally inRas-like protein polypeptides are described in basic texts, detailedmonographs, and the research literature, and they are well known tothose of skill in the art (some of these features are identified in FIG.2).

[0054] Known modifications include, but are not limited to, acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent crosslinks, formation of cystine, formation ofpyroglutamate, formylation, gamma carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination.

[0055] Such modifications are well known to those of skill in the artand have been described in great detail in the scientific literature.Several particularly common modifications, glycosylation, lipidattachment, sulfation, gamma-carboxylation of glutamic acid residues,hydroxylation and ADP-ribosylation, for instance, are described in mostbasic texts, such as Proteins—Structure and Molecular Properties, 2ndEd., T. E. Creighton, W. H. Freeman and Company, New York (1993). Manydetailed reviews are available on this subject, such as by Wold, F.,Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed.,Academic Press, New York 1-12 (1983); Seifter et al. (Meth. Enzymol 182:626-646 (1990)) and Rattan et aL (Ann. N. Y Acad. Sci. 663:48-62(1992)).

[0056] Accordingly, the Ras-like protein polypeptides of the presentinvention also encompass derivatives or analogs in which a substitutedamino acid residue is not one encoded by the genetic code, in which asubstituent group is included, in which the mature Ras-like proteinpolypeptide is fused with another compound, such as a compound toincrease the half-life of the Ras-like protein polypeptide (for example,polyethylene glycol), or in which the additional amino acids are fusedto the mature Ras-like protein polypeptide, such as a leader orsecretory sequence or a sequence for purification of the mature Ras-likeprotein polypeptide, or a pro-protein sequence.

Protein/Peptide Uses

[0057] The proteins of the present invention can be used in assays todetermine the biological activity of the protein, including in a panelof multiple proteins for high-throughput screening; to raise antibodiesor to elicit another immune response; as a reagent (including thelabeled reagent) in assays designed to quantitatively determine levelsof the protein (or its ligand or receptor) in biological fluids; and asmarkers for tissues in which the corresponding protein is preferentiallyexpressed (either constitutively or at a particular stage of tissuedifferentiation or development or in a disease state). Where the proteinbinds or potentially binds to another protein (such as, for example, ina receptor-ligand interaction), the protein can be used to identify thebinding partner so as to develop a system to identify inhibitors of thebinding interaction. Any or all of these research utilities are capableof being developed into reagent grade or kit format forcommercialization as research products.

[0058] Methods for performing the uses listed above are well known tothose skilled in the art. References disclosing such methods include“Molecular Cloning: A Laboratory Manual”, 2d ed., Cold Spring HarborLaboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds.,1989, and “Methods in Enzymology: Guide to Molecular CloningTechniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.

[0059] The potential uses of the peptides of the present invention arebased primarily on the source of the protein as well as the class/actionof the protein. For example, Ras-like proteins isolated from humans andtheir human/mammalian orthologs serve as targets for identifying agentsfor use in mammalian therapeutic applications, e.g. a human drug,particularly in modulating a biological or pathological response in acell or tissue that expresses the Ras-like protein. Experimental data asprovided in FIG. 1 indicates that Ras-like proteins of the presentinvention are expressed in humans in B-cells from tonsils, adrenalcortex carcinomas, Jurkat T-cells, prostate adenocarcinomas, lung(including normal and small cell carcinoma), marrow, and placenta, asindicated by virtual northern blot analysis. In addition, PCR-basedtissue screening panels indicate expression in human leukocytes. A largepercentage of pharmaceutical agents are being developed that modulatethe activity of Ras-like proteins, particularly members of the GEFsubfamily (see Background of the Invention). The structural andfunctional information provided in the Background and Figures providespecific and substantial uses for the molecules of the presentinvention, particularly in combination with the expression informationprovided in FIG. 1. Experimental data as provided in FIG. 1 indicatesexpression in humans in B-cells from tonsils, adrenal cortex carcinomas,Jurkat T-cells, prostate adenocarcinomas, lung (including normal andsmall cell carcinoma), marrow, placenta, and leukocytes. Such uses canreadily be determined using the information provided herein, that whichis known in the art, and routine experimentation.

[0060] The proteins of the present invention (including variants andfragments that may have been disclosed prior to the present invention)are useful for biological assays related to Ras-like proteins that arerelated to members of the GEF subfamily. Such assays involve any of theknown Ras-like protein functions or activities or properties useful fordiagnosis and treatment of Ras-like protein-related conditions that arespecific for the subfamily of Ras-like proteins that the one of thepresent invention belongs to, particularly in cells and tissues thatexpress the Ras-like protein. Experimental data as provided in FIG. 1indicates that Ras-like proteins of the present invention are expressedin humans in B-cells from tonsils, adrenal cortex carcinomas, JurkatT-cells, prostate adenocarcinomas, lung (including normal and small cellcarcinoma), marrow, and placenta, as indicated by virtual northern blotanalysis. In addition, PCR-based tissue screening panels indicateexpression in human leukocytes.

[0061] The proteins of the present invention are also useful in drugscreening assays, in cell-based or cell-free systems. Cell-based systemscan be native, i.e., cells that normally express the Ras-like protein,as a biopsy or expanded in cell culture. Experimental data as providedin FIG. 1 indicates expression in humans in B-cells from tonsils,adrenal cortex carcinomas, Jurkat T-cells, prostate adenocarcinomas,lung (including normal and small cell carcinoma), marrow, placenta, andleukocytes. In an alternate embodiment, cell-based assays involverecombinant host cells expressing the Ras-like protein.

[0062] The polypeptides can be used to identify compounds that modulateRas-like protein activity. Both the Ras-like protein of the presentinvention and appropriate variants and fragments can be used inhigh-throughput screens to assay candidate compounds for the ability tobind to the Ras-like protein. These compounds can be further screenedagainst a functional Ras-like protein to determine the effect of thecompound on the Ras-like protein activity. Further, these compounds canbe tested in animal or invertebrate systems to determineactivity/effectiveness. Compounds can be identified that activate(agonist) or inactivate (antagonist) the Ras-like protein to a desireddegree.

[0063] Therefore, in one embodiment, GEF or a fragment or derivativethereof may be administered to a subject to prevent or treat a disorderassociated with an increase in apoptosis. Such disorders include, butare not limited to, AIDS and other infectious or geneticimmunodeficiencies, neurodegenerative diseases such as Alzheimer'sdisease, Parkinson's disease, amyotrophic lateral sclerosis, retinitispigmentosa, and cerebellar degeneration, myelodysplastic syndromes suchas aplastic anemia, ischemic injuries such as myocardial infarction,stroke, and reperfusion injury, toxin-induced diseases such asalcohol-induced liver damage, cirrhosis, and lathyrism, wasting diseasessuch as cachexia, viral infections such as those caused by hepatitis Band C, and osteoporosis.

[0064] In another embodiment, a pharmaceutical composition comprisingGEF may be administered to a subject to prevent or treat a disorderassociated with increased apoptosis including, but not limited to, thoselisted above.

[0065] In still another embodiment, an agonist which is specific for GEFmay be administered to prevent or treat a disorder associated withincreased apoptosis including, but not limited to, those listed above.

[0066] In a further embodiment, a vector capable of expressing GEF, or afragment or a derivative thereof, may be used to prevent or treat adisorder associated with increased apoptosis including, but not limitedto, those listed above.

[0067] In cancer, where GEF promotes cell proliferation, it is desirableto decrease its activity. Therefore, in one embodiment, an antagonist ofGEF may be administered to a subject to prevent or treat cancerincluding, but not limited to, adenocarcinoma, leukemia, lymphoma,melanoma, myeloma, sarcoma, and teratocarcinoma, and, in particular,cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast,cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney,liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate,salivary glands, skin, spleen, testis, thymus, thyroid, and uterus. Inone aspect, an antibody specific for GEF may be used directly as anantagonist, or indirectly as a targeting or delivery mechanism forbringing a pharmaceutical agent to cells or tissue which express GEF.

[0068] In another embodiment, a vector expressing the complement of thepolynucleotide encoding GEF may be administered to a subject to preventor treat a cancer including, but not limited to, the types of cancerlisted above.

[0069] In inflammation, where GEF promotes cell proliferation, it isdesirable to decrease its activity. Therefore, in one embodiment, anantagonist of GEF may be administered to a subject to prevent or treatan inflammation. Disorders associated with inflammation include, but arenot limited to, Addison's disease, adult respiratory distress syndrome,allergies, anemia, asthma, atherosclerosis, bronchitis, cholecystitis,Crohn's disease, ulcerative colitis, atopic dermatitis, dermatomyositis,diabetes mellitus, emphysema, atrophic gastritis, glomerulonephritis,gout, Graves' disease, hypereosinophilia, irritable bowel syndrome,lupus erythematosus, multiple sclerosis, myasthenia gravis, myocardialor pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,polymyositis, rheumatoid arthritis, scleroderma, Sjogren's syndrome, andautoimmune thyroiditis; complications of cancer, hemodialysis,extracorporeal circulation; viral, bacterial, fungal, parasitic,protozoal, and helminthic infections and trauma. In one aspect, anantibody specific for GEF may be used directly as an antagonist, orindirectly as a targeting or delivery mechanism for bringing apharmaceutical agent to cells or tissue which express GEF.

[0070] Further, the Ras-like protein polypeptides can be used to screena compound for the ability to stimulate or inhibit interaction betweenthe Ras-like protein and a molecule that normally interacts with theRas-like protein, e.g. a ligand or a component of the signal pathwaythat the Ras-like protein normally interacts. Such assays typicallyinclude the steps of combining the Ras-like protein with a candidatecompound under conditions that allow the Ras-like protein, or fragment,to interact with the target molecule, and to detect the formation of acomplex between the protein and the target or to detect the biochemicalconsequence of the interaction with the Ras-like protein and the target,such as any of the associated effects of signal transduction.

[0071] Candidate compounds include, for example, 1) peptides such assoluble peptides, including Ig-tailed fusion peptides and members ofrandom peptide libraries (see, e.g., Lam et al., Nature 354:82-84(1991); Houghten et al., Nature 354:84-86 (1991)) and combinatorialchemistry-derived molecular libraries made of D- and/or L-configurationamino acids; 2) phosphopeptides (e.g., members of random and partiallydegenerate, directed phosphopeptide libraries, see, e.g., Songyang etal., Cell 72:767-778 (1993));3) antibodies (e.g., polyclonal,monoclonal, humanized, anti-idiotypic, chimeric, and single chainantibodies as well as Fab, F(ab′)₂, Fab expression library fragments,and epitope-binding fragments of antibodies); and 4) small organic andinorganic molecules (e.g., molecules obtained from combinatorial andnatural product libraries). (Hodgson, Bio/technology, Sep. 10, 1992(9);973-80).

[0072] One candidate compound is a soluble fragment of the Ras-likeprotein that competes for ligand binding. Other candidate compoundsinclude mutant Ras-like proteins or appropriate fragments containingmutations that affect Ras-like protein function and thus compete forligand. Accordingly, a fragment that competes for ligand, for examplewith a higher affinity, or a fragment that binds ligand but does notallow release, is within the scope of the invention.

[0073] The invention further includes other end point assays to identifycompounds that modulate (stimulate or inhibit) Ras-like proteinactivity. The assays typically involve an assay of events in theRas-like protein mediated signal transduction pathway that indicateRas-like protein activity. Thus, the phosphorylation of a protein/ligandtarget, the expression of genes that are up- or down-regulated inresponse to the Ras-like protein dependent signal cascade can beassayed. In one embodiment, the regulatory region of such genes can beoperably linked to a marker that is easily detectable, such asluciferase. Alternatively, phosphorylation of the Ras-like protein, or aRas-like protein target, could also be measured.

[0074] Any of the biological or biochemical functions mediated by theRas-like protein can be used as an endpoint assay. These include all ofthe biochemical or biochemical/biological events described herein, inthe references cited herein, incorporated by reference for theseendpoint assay targets, and other functions known to those of ordinaryskill in the art.

[0075] Binding and/or activating compounds can also be screened by usingchimeric Ras- like proteins in which any of the protein's domains, orparts thereof, can be replaced by heterologous domains or subregions.Accordingly, a different set of signal transduction components isavailable as an end-point assay for activation. This allows for assaysto be performed in other than the specific host cell from which theRas-like protein is derived.

[0076] The Ras-like protein polypeptide of the present invention is alsouseful in competition binding assays in methods designed to discovercompounds that interact with the Ras-like protein. Thus, a compound isexposed to a Ras-like protein polypeptide under conditions that allowthe compound to bind or to otherwise interact with the polypeptide.Soluble Ras-like protein polypeptide is also added to the mixture. Ifthe test compound interacts with the soluble Ras-like proteinpolypeptide, it decreases the amount of complex formed or activity fromthe Ras-like protein target. This type of assay is particularly usefulin cases in which compounds are sought that interact with specificregions of the Ras-like protein. Thus, the soluble polypeptide thatcompetes with the target Ras-like protein region is designed to containpeptide sequences corresponding to the region of interest.

[0077] To perform cell free drug screening assays, it is sometimesdesirable to immobilize either the Ras-like protein, or fragment, or itstarget molecule to facilitate separation of complexes from uncomplexedforms of one or both of the proteins, as well as to accommodateautomation of the assay.

[0078] Techniques for immobilizing proteins on matrices can be used inthe drug screening assays. In one embodiment, a fusion protein can beprovided which adds a domain that allows the protein to be bound to amatrix. For example, glutathione-S-transferase/15625 fusion proteins canbe adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis,Mo.) or glutathione derivatized microtitre plates, which are thencombined with the cell lysates (e.g., ³⁵S-labeled) and the candidatecompound, and the mixture incubated under conditions conducive tocomplex formation (e.g., at physiological conditions for salt and pH).Following incubation, the beads are washed to remove any unbound label,and the matrix immobilized and radiolabel determined directly, or in thesupernatant after the complexes are dissociated. Alternatively, thecomplexes can be dissociated from the matrix, separated by SDS-PAGE, andthe level of Ras-like protein-binding protein found in the bead fractionquantitated from the gel using standard electrophoretic techniques. Forexample, either the polypeptide or its target molecule can beimmobilized utilizing conjugation of biotin and streptavidin withtechniques well known in the art. Alternatively, antibodies reactivewith the protein but which do not interfere with binding of the proteinto its target molecule can be derivatized to the wells of the plate, andthe protein trapped in the wells by antibody conjugation. Preparationsof a Ras-like protein-binding protein and a candidate compound areincubated in the Ras-like protein-presenting wells and the amount ofcomplex trapped in the well can be quantitated. Methods for detectingsuch complexes, in addition to those described above for theGST-immobilized complexes, include immunodetection of complexes usingantibodies reactive with the Ras-ike protein target molecule, or whichare reactive with Ras-like protein and compete with the target molecule,as well as enzyme-linked assays which rely on detecting an enzymaticactivity associated with the target molecule.

[0079] Agents that modulate one of the Ras-like proteins of the presentinvention can be identified using one or more of the above assays, aloneor in combination. It is generally preferable to use a cell-based orcell free system first and then confirm activity in an animal/insectmodel system. Such model systems are well known in the art and canreadily be employed in this context.

[0080] Modulators of Ras-like protein activity identified according tothese drug screening assays can be used to treat a subject with adisorder mediated by the Ras-like protein associated pathway, bytreating cells that express the Ras-like protein. Experimental data asprovided in FIG. 1 indicates expression in humans in B-cells fromtonsils, adrenal cortex carcinomas, Jurkat T-cells, prostateadenocarcinomas, lung (including normal and small cell carcinoma),marrow, placenta, and leukocytes. These methods of treatment include thesteps of administering the modulators of protein activity in apharmaceutical composition as described herein, to a subject in need ofsuch treatment.

[0081] In yet another aspect of the invention, the Ras-like proteins canbe used as “bait proteins” in a two-hybrid assay or three-hybrid assay(see, e.g., U.S. Pat. No. 5,283,317; Zervos et al., Cell 72:223-232(1993); Madura et al., J. Biol. Chem. 268:12046-12054 (1993); Bartel etal., Biotechniques 14:920-924 (1993); Iwabuchi et al., Oncogene8:1693-1696 (1993); and Brent WO94/10300), to identify other proteinsthat bind to or interact with the Ras-like protein and are involved inRas-like protein activity. Such Ras-like protein-binding proteins arealso likely to be involved in the propagation of signals by the Ras-likeproteins or Ras-like protein targets as, for example, downstreamelements of a Ras-like protein-mediated signaling pathway, e.g., a painsignaling pathway. Alternatively, such Ras-like protein-binding proteinsare likely to be Ras-like protein inhibitors.

[0082] The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for a Ras-like proteinis fused to a gene encoding the DNA binding domain of a knowntranscription factor (e.g., GAL-4). In the other construct, a DNAsequence, from a library of DNA sequences, that encodes an unidentifiedprotein (“prey” or “sample”) is fused to a gene that codes for theactivation domain of the known transcription factor. If the “bait” andthe “prey” proteins are able to interact, in vivo, forming a Ras-likeprotein-dependent complex, the DNA-binding and activation domains of thetranscription factor are brought into close proximity. This proximityallows transcription of a reporter gene (e.g., LacZ) which is operablylinked to a transcriptional regulatory site responsive to thetranscription factor. Expression of the reporter gene can be detectedand cell colonies containing the functional transcription factor can beisolated and used to obtain the cloned gene which encodes the proteinwhich interacts with the Ras-like protein.

[0083] This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate animal model. For example, an agent identified asdescribed herein (e.g., a Ras-like protein modulating agent, anantisense Ras-like protein nucleic acid molecule, a Ras-likeprotein-specific antibody, or a Ras-like protein-binding partner) can beused in an animal or insect model to determine the efficacy, toxicity,or side effects of treatment with such an agent. Alternatively, an agentidentified as described herein can be used in an animal or insect modelto determine the mechanism of action of such an agent. Furthermore, thisinvention pertains to uses of novel agents identified by theabove-described screening assays for treatments as described herein.

[0084] The Ras-like proteins of the present invention are also useful toprovide a target for diagnosing a disease or predisposition to a diseasemediated by the peptide, Accordingly, the invention provides methods fordetecting the presence, or levels of, the protein (or encoding mRNA) ina cell, tissue, or organism. Experimental data as provided in FIG. 1indicates expression in humans in B-cells from tonsils, adrenal cortexcarcinomas, Jurkat T-cells, prostate adenocarcinomas, lung (includingnormal and small cell carcinoma), marrow, placenta, and leukocytes. Themethod involves contacting a biological sample with a compound capableof interacting with the receptor protein such that the interaction canbe detected. Such an assay can be provided in a single detection formator a multi-detection format such as an antibody chip array.

[0085] One agent for detecting a protein in a sample is an antibodycapable of selectively binding to protein. A biological sample includestissues, cells and biological fluids isolated from a subject, as well astissues, cells, and fluids present within a subject.

[0086] The peptides also are useful to provide a target for diagnosing adisease or predisposition to a disease mediated by the peptide,Accordingly, the invention provides methods for detecting the presence,or levels of, the protein in a cell, tissue, or organism. The methodinvolves contacting a biological sample with a compound capable ofinteracting with the receptor protein such that the interaction can bedetected.

[0087] The peptides of the present invention also provide targets fordiagnosing active disease, or predisposition to a disease, in a patienthaving a variant peptide. Thus, the peptide can be isolated from abiological sample and assayed for the presence of a genetic mutationthat results in translation of an aberrant peptide. This includes aminoacid substitution, deletion, insertion, rearrangement, (as the result ofaberrant splicing events), and inappropriate post-translationalmodification. Analytic methods include altered electrophoretic mobility,altered tryptic peptide digest, altered receptor activity in cell-basedor cell-free assay, alteration in ligand or antibody-binding pattern,altered isoelectric point, direct amino acid sequencing, and any otherof the known assay techniques useful for detecting mutations in aprotein. Such an assay can be provided in a single detection format or amulti-detection format such as an antibody chip array.

[0088] In vitro techniques for detection of peptide include enzymelinked immunosorbent assays (ELISAs), Western blots,immunoprecipitations, and immunofluorescence using a detection reagents,such as an antibody or protein binding agent. Alternatively, the peptidecan be detected in vivo in a subject by introducing into the subject alabeled anti-peptide antibody. For example, the antibody can be labeledwith a radioactive marker whose presence and location in a subject canbe detected by standard imaging techniques. Particularly useful aremethods that detect the allelic variant of a peptide expressed in asubject and methods which detect fragments of a peptide in a sample.

[0089] The peptides are also useful in pharmacogenomic analysis.Pharmacogenomics deal with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, e.g., Eichelbaum, M. (Clin. Exp.Pharmacol. Physiol. 23(10-11) :983-985 (1996)), and Linder, M. W. (Clin.Chem. 43(2):254-266 (1997)). The clinical outcomes of these variationsresult in severe toxicity of therapeutic drugs in certain individuals ortherapeutic failure of drugs in certain individuals as a result ofindividual variation in metabolism. Thus, the genotype of the individualcan determine the way a therapeutic compound acts on the body or the waythe body metabolizes the compound. Further, the activity of drugmetabolizing enzymes effects both the intensity and duration of drugaction. Thus, the pharmacogenomics of the individual permit theselection of effective compounds and effective dosages of such compoundsfor prophylactic or therapeutic treatment based on the individual'sgenotype. The discovery of genetic polymorphisms in some drugmetabolizing enzymes has explained why some patients do not obtain theexpected drug effects, show an exaggerated drug effect, or experienceserious toxicity from standard drug dosages. Polymorphisms can beexpressed in the phenotype of the extensive metabolizer and thephenotype of the poor metabolizer. Accordingly, genetic polymorphism maylead to allelic protein variants of the receptor protein in which one ormore of the receptor functions in one population is different from thosein another population. The peptides thus allow a target to ascertain agenetic predisposition that can affect treatment modality. Thus, in aligand-based treatment, polymorphism may give rise to amino terminalextracellular domains and/or other ligand-binding regions that are moreor less active in ligand binding, and receptor activation. Accordingly,ligand dosage would necessarily be modified to maximize the therapeuticeffect within a given population containing a polymorphism. As analternative to genotyping, specific polymorphic peptides could beidentified.

[0090] The peptides are also useful for treating a disordercharacterized by an absence of, inappropriate, or unwanted expression ofthe protein. Experimental data as provided in FIG. 1 indicatesexpression in humans in B-cells from tonsils, adrenal cortex carcinomas,Jurkat T-cells, prostate adenocarcinomas, lung (including normal andsmall cell carcinoma), marrow, placenta, and leukocytes. Accordingly,methods for treatment include the use of the Ras-like protein orfragments.

Antibodies

[0091] The invention also provides antibodies that selectively bind toone of the peptides of the present invention, a protein comprising sucha peptide, as well as variants and fragments thereof. As used herein, anantibody selectively binds a target peptide when it binds the targetpeptide and does not significantly bind to unrelated proteins. Anantibody is still considered to selectively bind a peptide even if italso binds to other proteins that are not substantially homologous withthe target peptide so long as such proteins share homology with afragment or domain of the peptide target of the antibody. In this case,it would be understood that antibody binding to the peptide is stillselective despite some degree of cross-reactivity.

[0092] As used herein, an antibody is defined in terms consistent withthat recognized within the art: they are multi-subunit proteins producedby a mammalian organism in response to an antigen challenge. Theantibodies of the present invention include polyclonal antibodies andmonoclonal antibodies, as well as fragments of such antibodies,including, but not limited to, Fab or F(ab′)₂, and Fv fragments.

[0093] Many methods are known for generating and/or identifyingantibodies to a given target peptide. Several such methods are describedby Harlow, Antibodies, Cold Spring Harbor Press, (1989).

[0094] In general, to generate antibodies, an isolated peptide is usedas an immunogen and is administered to a mammalian organism, such as arat, rabbit or mouse. The full-length protein, an antigenic peptidefragment or a fusion protein can be used. Particularly importantfragments are those covering functional domains, such as the domainsidentified in FIG. 2, and domain of sequence homology or divergenceamongst the family, such as those that can readily be identified usingprotein alignment methods and as presented in the Figures.

[0095] Antibodies are preferably prepared from regions or discretefragments of the Ras-like proteins. Antibodies can be prepared from anyregion of the peptide as described herein. However, preferred regionswill include those involved in function/activity and/or receptor/bindingpartner interaction. FIG. 2 can be used to identify particularlyimportant regions while sequence alignment can be used to identifyconserved and unique sequence fragments.

[0096] An antigenic fragment will typically comprise at least 8contiguous amino acid residues. The antigenic peptide can comprise,however, at least 10, 12, 14, 16 or more amino acid residues. Suchfragments can be selected on a physical property, such as fragmentscorrespond to regions that are located on the surface of the protein,e.g., hydrophilic regions or can be selected based on sequenceuniqueness (see FIG. 2).

[0097] Detection of an antibody of the present invention can befacilitated by coupling (i.e., physically linking) the antibody to adetectable substance. Examples of detectable substances include variousenzymes, prosthetic groups, fluorescent materials, luminescentmaterials, bioluminescent materials, and radioactive materials. Examplesof suitable enzymes include horseradish peroxidase, alkalinephosphatase, β-galactosidase, or acetylcholinesterase; examples ofsuitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S,or ³H.

Antibody Uses

[0098] The antibodies can be used to isolate one of the proteins of thepresent invention by standard techniques, such as affinitychromatography or immunoprecipitation. The antibodies can facilitate thepurification of the natural protein from cells and recombinantlyproduced protein expressed in host cells. In addition, such antibodiesare useful to detect the presence of one of the proteins of the presentinvention in cells or tissues to determine the pattern of expression ofthe protein among various tissues in an organism and over the course ofnormal development. Experimental data as provided in FIG. 1 indicatesthat Ras-like proteins of the present invention are expressed in humansin B-cells from tonsils, adrenal cortex carcinomas, Jurkat T-cells,prostate adenocarcinomas, lung (including normal and small cellcarcinoma), marrow, and placenta, as indicated by virtual northern blotanalysis. In addition, PCR-based tissue screening panels indicateexpression in human leukocytes. Further, such antibodies can be used todetect protein in situ, in vitro, or in a cell lysate or supernatant inorder to evaluate the abundance and pattern of expression. Also, suchantibodies can be used to assess abnormal tissue distribution orabnormal expression during development. Antibody detection ofcirculating fragments of the full-length protein can be used to identifyturnover.

[0099] Further, the antibodies can be used to assess expression indisease states such as in active stages of the disease or in anindividual with a predisposition toward disease related to the protein'sfunction. When a disorder is caused by an inappropriate tissuedistribution, developmental expression, level of expression of theprotein, or expressed/processed form, the antibody can be preparedagainst the normal protein. Experimental data as provided in FIG. 1indicates expression in humans in B-cells from tonsils, adrenal cortexcarcinomas, Jurkat T-cells, prostate adenocarcinomas, lung (includingnormal and small cell carcinoma), marrow, placenta, and leukocytes. If adisorder is characterized by a specific mutation in the protein,antibodies specific for this mutant protein can be used to assay for thepresence of the specific mutant protein.

[0100] The antibodies can also be used to assess normal and aberrantsubcellular localization of cells in the various tissues in an organism.Experimental data as provided in FIG. 1 indicates expression in humansin B-cells from tonsils, adrenal cortex carcinomas, Jurkat T-cells,prostate adenocarcinomas, lung (including normal and small cellcarcinoma), marrow, placenta, and leukocytes. The diagnostic uses can beapplied, not only in genetic testing, but also in monitoring a treatmentmodality. Accordingly, where treatment is ultimately aimed at correctingexpression level or the presence of aberrant sequence and aberranttissue distribution or developmental expression, antibodies directedagainst the or relevant fragments can be used to monitor therapeuticefficacy.

[0101] Additionally, antibodies are useful in pharmacogenomic analysis.Thus, antibodies prepared against polymorphic proteins can be used toidentify individuals that require modified treatment modalities. Theantibodies are also useful as diagnostic tools as an immunologicalmarker for aberrant protein analyzed by electrophoretic mobility,isoelectric point, tryptic peptide digest, and other physical assaysknown to those in the art.

[0102] The antibodies are also useful for tissue typing. Experimentaldata as provided in FIG. 1 indicates expression in humans in B-cellsfrom tonsils, adrenal cortex carcinomas, Jurkat T-cells, prostateadenocarcinomas, lung (including normal and small cell carcinoma),marrow, placenta, and leukocytes. Thus, where a specific protein hasbeen correlated with expression in a specific tissue, antibodies thatare specific for this protein can be used to identify a tissue type.

[0103] The antibodies are also useful for inhibiting protein function,for example, blocking the binding of the Ras-like protein to a bindingpartner such as a substrate. These uses can also be applied in atherapeutic context in which treatment involves inhibiting the protein'sfunction. An antibody can be used, for example, to block binding, thusmodulating (agonizing or antagonizing) the peptides activity. Antibodiescan be prepared against specific fragments containing sites required forfunction or against intact protein that is associated with a cell orcell membrane. See FIG. 2 for structural information relating to theproteins of the present invention.

[0104] The invention also encompasses kits for using antibodies todetect the presence of a protein in a biological sample. The kit cancomprise antibodies such as a labeled or labelable antibody and acompound or agent for detecting protein in a biological sample; meansfor determining the amount of protein in the sample; means for comparingthe amount of protein in the sample with a standard; and instructionsfor use.

Nucleic Acid Molecules

[0105] The present invention further provides isolated nucleic acidmolecules that encode a Ras-like protein polypeptide of the presentinvention. Such nucleic acid molecules will consist of, consistessentially of, or comprise a nucleotide sequence that encodes one ofthe Ras-like protein polypeptides of the present invention, an allelicvariant thereof, or an ortholog or paralog thereof.

[0106] As used herein, an “isolated” nucleic acid molecule is one thatis separated from other nucleic acid present in the natural source ofthe nucleic acid. Preferably, an “isolated” nucleic acid is free ofsequences which naturally flank the nucleic acid (i.e., sequenceslocated at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA ofthe organism from which the nucleic acid is derived. However, there canbe some flanking nucleotide sequences, for example up to about 5KB,particularly contiguous peptide encoding sequences and peptide encodingsequences within the same gene but separated by introns in the genomicsequence. The important point is that the nucleic acid is isolated fromremote and unimportant flanking sequences such that it can be subjectedto the specific manipulations described herein such as recombinantexpression, preparation of probes and primers, and other uses specificto the nucleic acid sequences.

[0107] Moreover, an “isolated” nucleic acid molecule, such as a cDNAmolecule, can be substantially free of other cellular material, orculture medium when produced by recombinant techniques, or chemicalprecursors or other chemicals when chemically synthesized. However, thenucleic acid molecule can be fused to other coding or regulatorysequences and still be considered isolated.

[0108] For example, recombinant DNA molecules contained in a vector areconsidered isolated. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells orpurified (partially or substantially) DNA molecules in solution.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofthe isolated DNA molecules of the present invention. Isolated nucleicacid molecules according to the present invention further include suchmolecules produced synthetically.

[0109] Accordingly, the present invention provides nucleic acidmolecules that consist of the nucleotide sequence shown in FIG. 1 or 3(SEQ ID NO:1, transcript sequence and SEQ ID NO:3, genomic sequence), orany nucleic acid molecule that encodes the protein provided in FIG. 2,SEQ ID NO:2. A nucleic acid molecule consists of a nucleotide sequencewhen the nucleotide sequence is the complete nucleotide sequence of thenucleic acid molecule. The present invention further provides nucleicacid molecules that consist essentially of the nucleotide sequence shownin FIG. 1 or 3 (SEQ ID NO:1, transcript sequence and SEQ ID NO:3,genomic sequence), or any nucleic acid molecule that encodes the proteinprovided in FIG. 2, SEQ ID NO:2. A nucleic acid molecule consistsessentially of a nucleotide sequence when such a nucleotide sequence ispresent with only a few additional nucleic acid residues in the finalnucleic acid molecule.

[0110] The present invention further provides nucleic acid moleculesthat comprise the nucleotide sequences shown in FIG. 1 or 3 (SEQ IDNO:1, transcript sequence and SEQ ID NO:3, genomic sequence), or anynucleic acid molecule that encodes the protein provided in FIG. 2, SEQID NO:2. A nucleic acid molecule comprises a nucleotide sequence whenthe nucleotide sequence is at least part of the final nucleotidesequence of the nucleic acid molecule. In such a fashion, the nucleicacid molecule can be only the nucleotide sequence or have additionalnucleic acid residues, such as nucleic acid residues that are naturallyassociated with it or heterologous nucleotide sequences. Such a nucleicacid molecule can have a few additional nucleotides or can comprisesseveral hundred or more additional nucleotides. A brief description ofhow various types of these nucleic acid molecules can be readilymade/isolated is provided below.

[0111] In FIGS. 1 and 3, both coding and non-coding sequences areprovided. Because of the source of the present invention, humans genomicsequence (FIG. 3) and cDNA/transcript sequences (FIG. 1), the nucleicacid molecules in the Figures will contain genomic intronic sequences,5′ and 3′ non-coding sequences, gene regulatory regions and non-codingintergenic sequences. In general such sequence features are either notedin FIGS. 1 and 3 or can readily be identified using computational toolsknown in the art. As discussed below, some of the non-coding regions,particularly gene regulatory elements such as promoters, are useful fora variety of purposes, e.g. control of heterologous gene expression,target for identifying gene activity modulating compounds, and areparticularly claimed as fragments of the genomic sequence providedherein.

[0112] Full-length genes may be cloned from known sequence using any oneof a number of methods known in the art. For example, a method whichemploys XL-PCR (Perkin-Elmer, Foster City, Calif.) to amplify longpieces of DNA may be used. Other methods for obtaining full-lengthsequences are well known in the art.

[0113] The isolated nucleic acid molecules can encode the mature proteinplus additional amino or carboxyl-terminal amino acids, or amino acidsinterior to the mature peptide (when the mature form has more than onepeptide chain, for instance). Such sequences may play a role inprocessing of a protein from precursor to a mature form, facilitateprotein trafficking, prolong or shorten protein half-life, or facilitatemanipulation of a protein for assay or production, among other things.As generally is the case in situ, the additional amino acids may beprocessed away from the mature protein by cellular enzymes.

[0114] As mentioned above, the isolated nucleic acid molecules include,but are not limited to, the sequence encoding the Ras-like proteinpolypeptide alone, the sequence encoding the mature peptide andadditional coding sequences, such as a leader or secretory sequence(e.g., a pre-pro or pro-protein sequence), the sequence encoding themature peptide, with or without the additional coding sequences, plusadditional non-coding sequences, for example introns and non-coding 5′and 3′ sequences such as transcribed but non-translated sequences thatplay a role in transcription, mRNA processing (including splicing andpolyadenylation signals), ribosome binding, and stability of mRNA. Inaddition, the nucleic acid molecule may be fused to a marker sequenceencoding, for example, a peptide that facilitates purification.

[0115] Isolated nucleic acid molecules can be in the form of RNA, suchas mRNA, or in the form of DNA, including cDNA and genomic DNA obtainedby cloning or produced by chemical synthetic techniques or by acombination thereof. The nucleic acid, especially DNA, can bedouble-stranded or single-stranded. Single-stranded nucleic acid can bethe coding strand (sense strand) or the non-coding strand (anti-sensestrand).

[0116] The invention further provides nucleic acid molecules that encodefragments of the peptides of the present invention and that encodeobvious variants of the Ras-like proteins of the present invention thatare described above. Such nucleic acid molecules may be naturallyoccurring, such as allelic variants (same locus), paralogs (differentlocus), and orthologs (different organism), or may be constructed byrecombinant DNA methods or by chemical synthesis. Such non-naturallyoccurring variants may be made by mutagenesis techniques, includingthose applied to nucleic acid molecules, cells, or whole organisms.Accordingly, as discussed above, the variants can contain nucleotidesubstitutions, deletions inversions, and/or insertions. Variation canoccur in either or both the coding and non-coding regions. Thevariations can produce both conservative and non-conservative amino acidsubstitutions.

[0117] The present invention further provides non-coding fragments ofthe nucleic acid molecules provided in the FIGS. 1 and 3. Preferrednon-coding fragments include, but are not limited to, promotersequences, enhancer sequences, gene modulating sequences, and genetermination sequences. Such fragments are useful in controllingheterologous gene expression and in developing screens to identifygene-modulating agents.

[0118] A fragment comprises a contiguous nucleotide sequence greaterthan 12 or more nucleotides. Further, a fragment could be at least 30,40, 50, 100 250, or 500 nucleotides in length. The length of thefragment will be based on its intended use. For example, the fragmentcan encode epitope-bearing regions of the peptide, or can be useful asDNA probes and primers. Such fragments can be isolated using the knownnucleotide sequence to synthesize an oligonucleotide probe. A labeledprobe can then be used to screen a cDNA library, genomic DNA library, ormRNA to isolate nucleic acid corresponding to the coding region.Further, primers can be used in PCR reactions to clone specific regionsof gene.

[0119] A probe/primer typically comprises substantially a purifiedoligonucleotide or oligonucleotide pair. The oligonucleotide typicallycomprises a region of nucleotide sequence that hybridizes understringent conditions to at least about 12, 20, 25, 40, 50, or moreconsecutive nucleotides.

[0120] Orthologs, homologs, and allelic variants can be identified usingmethods well known in the art. As described in the Peptide Section,these variants comprise a nucleotide sequence encoding a peptide that istypically 60-70%, 70-80%, 80-90%, and more typically at least about90-95% or more homologous to the nucleotide sequence shown in the Figuresheets or a fragment of this sequence. Such nucleic acid molecules canreadily be identified as being able to hybridize under moderate tostringent conditions, to the nucleotide sequence shown in the Figuresheets or a fragment of the sequence. The gene encoding the novelRas-like protein of the present invention is located on a genomecomponent that has been mapped to human chromosome 9 (as indicated inFIG. 3), which is supported by multiple lines of evidence, such as STSand BAC map data.

[0121] As used herein, the term “hybridizes under stringent conditions”is intended to describe conditions for hybridization and washing underwhich nucleotide sequences encoding a peptide at least 60-70% homologousto each other typically remain hybridized to each other. The conditionscan be such that sequences at least about 60%, at least about 70%, or atleast about 80% or more homologous to each other typically remainhybridized to each other. Such stringent conditions are known to thoseskilled in the art and can be found in Current Protocols in MolecularBiology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. One example ofstringent hybridization conditions are hybridization in 6× sodiumchloride/sodium citrate (SSC) at about 45C, followed by one or morewashes in 0.2×SSC, 0.1% SDS at 50-65° C. Examples of moderate to lowstringency hybridization conditions are well known in the art.

Nucleic Acid Molecule Uses

[0122] The nucleic acid molecules of the present invention are usefulfor probes, primers, chemical intermediates, and in biological assays.The nucleic acid molecules are useful as a hybridization probe formessenger RNA, transcript/cDNA and genomic DNA to isolate full-lengthcDNA and genomic clones encoding the peptide described in FIG. 2 and toisolate cDNA and genomic clones that correspond to variants (alleles,orthologs, etc.) producing the same or related peptides shown in FIG. 2.

[0123] The probe can correspond to any sequence along the entire lengthof the nucleic acid molecules provided in the Figures. Accordingly, itcould be derived from 5′ noncoding regions, the coding region, and 3′noncoding regions. However, as discussed, fragments are not to beconstrued as those, which may encompass fragments disclosed prior to thepresent invention.

[0124] The nucleic acid molecules are also useful as primers for PCR toamplify any given region of a nucleic acid molecule and are useful tosynthesize antisense molecules of desired length and sequence.

[0125] The nucleic acid molecules are also useful for constructingrecombinant vectors. Such vectors include expression vectors thatexpress a portion of, or all of, the peptide sequences. Vectors alsoinclude insertion vectors, used to integrate into another nucleic acidmolecule sequence, such as into the cellular genome, to alter in situexpression of a gene and/or gene product. For example, an endogenouscoding sequence can be replaced via homologous recombination with all orpart of the coding region containing one or more specifically introducedmutations.

[0126] The nucleic acid molecules are also useful for expressingantigenic portions of the proteins.

[0127] The nucleic acid molecules are also useful as probes fordetermining the chromosomal positions of the nucleic acid molecules bymeans of in situ hybridization methods. The gene encoding the novelRas-like protein of the present invention is located on a genomecomponent that has been mapped to human chromosome 9 (as indicated inFIG. 3), which is supported by multiple lines of evidence, such as STSand BAC map data.

[0128] The nucleic acid molecules are also useful in making vectorscontaining the gene regulatory regions of the nucleic acid molecules ofthe present invention.

[0129] The nucleic acid molecules are also useful for designingribozymes corresponding to all, or a part, of the mRNA produced from thenucleic acid molecules described herein.

[0130] The nucleic acid molecules are also useful for constructing hostcells expressing a part, or all, of the nucleic acid molecules andpeptides. Moreover, the nucleic acid molecules are useful forconstructing transgenic animals wherein a homolog of the nucleic acidmolecule has been “knocked-out” of the animal's genome.

[0131] The nucleic acid molecules are also useful for constructingtransgenic animals expressing all, or a part, of the nucleic acidmolecules and peptides.

[0132] The nucleic acid molecules are also useful for making vectorsthat express part, or all, of the peptides.

[0133] The nucleic acid molecules are also useful as hybridizationprobes for determining the presence, level, form, and distribution ofnucleic acid expression. Experimental data as provided in FIG. 1indicates that Ras-like proteins of the present invention are expressedin humans in B-cells from tonsils, adrenal cortex carcinomas, JurkatT-cells, prostate adenocarcinomas, lung (including normal and small cellcarcinoma), marrow, and placenta, as indicated by virtual northern blotanalysis. In addition, PCR-based tissue screening panels indicateexpression in human leukocytes. Accordingly, the probes can be used todetect the presence of, or to determine levels of, a specific nucleicacid molecule in cells, tissues, and in organisms. The nucleic acidwhose level is determined can be DNA or RNA. Accordingly, probescorresponding to the peptides described herein can be used to assessexpression and/or gene copy number in a given cell, tissue, or organism.These uses are relevant for diagnosis of disorders involving an increaseor decrease in Ras-like protein expression relative to normal results.

[0134] In vitro techniques for detection of mRNA include Northernhybridizations and in situ hybridizations. In vitro techniques fordetecting DNA include Southern hybridizations and in situ hybridization.

[0135] Probes can be used as a part of a diagnostic test kit foridentifying cells or tissues that express a Ras-like protein, such as bymeasuring a level of a receptor-encoding nucleic acid in a sample ofcells from a subject e.g., mRNA or genomic DNA, or determining if areceptor gene has been mutated. Experimental data as provided in FIG. 1indicates that Ras-like proteins of the present invention are expressedin humans in B-cells from tonsils, adrenal cortex carcinomas, JurkatT-cells, prostate adenocarcinomas, lung (including normal and small cellcarcinoma), marrow, and placenta, as indicated by virtual northern blotanalysis. In addition, PCR-based tissue screening panels indicateexpression in human leukocytes.

[0136] Nucleic acid expression assays are useful for drug screening toidentify compounds that modulate Ras-like protein nucleic acidexpression.

[0137] The invention thus provides a method for identifying a compoundthat can be used to treat a disorder associated with nucleic acidexpression of the Ras-like protein gene, particularly biological andpathological processes that are mediated by the Ras-like protein incells and tissues that express it. Experimental data as provided in FIG.1 indicates expression in humans in B-cells from tonsils, adrenal cortexcarcinomas, Jurkat T-cells, prostate adenocarcinomas, lung (includingnormal and small cell carcinoma), marrow, placenta, and leukocytes. Themethod typically includes assaying the ability of the compound tomodulate the expression of the Ras-like protein nucleic acid and thusidentifying a compound that can be used to treat a disordercharacterized by undesired Ras-like protein nucleic acid expression. Theassays can be performed in cell-based and cell-free systems. Cell-basedassays include cells naturally expressing the Ras-like protein nucleicacid or recombinant cells genetically engineered to express specificnucleic acid sequences.

[0138] The assay for Ras-like protein nucleic acid expression caninvolve direct assay of nucleic acid levels, such as mRNA levels, or oncollateral compounds involved in the signal pathway. Further, theexpression of genes that are up- or down-regulated in response to theRas-like protein signal pathway can also be assayed. In this embodimentthe regulatory regions of these genes can be operably linked to areporter gene such as luciferase.

[0139] Thus, modulators of Ras-like protein gene expression can beidentified in a method wherein a cell is contacted with a candidatecompound and the expression of mRNA determined. The level of expressionof Ras-like protein mRNA in the presence of the candidate compound iscompared to the level of expression of Ras-like protein mRNA in theabsence of the candidate compound. The candidate compound can then beidentified as a modulator of nucleic acid expression based on thiscomparison and be used, for example to treat a disorder characterized byaberrant nucleic acid expression. When expression of mRNA isstatistically significantly greater in the presence of the candidatecompound than in its absence, the candidate compound is identified as astimulator of nucleic acid expression. When nucleic acid expression isstatistically significantly less in the presence of the candidatecompound than in its absence, the candidate compound is identified as aninhibitor of nucleic acid expression.

[0140] The invention further provides methods of treatment, with thenucleic acid as a target, using a compound identified through drugscreening as a gene modulator to modulate Ras-like protein nucleic acidexpression in cells and tissues that express the Ras-like protein.Experimental data as provided in FIG. 1 indicates that Ras-like proteinsof the present invention are expressed in humans in B-cells fromtonsils, adrenal cortex carcinomas, Jurkat T-cells, prostateadenocarcinomas, lung (including normal and small cell carcinoma),marrow, and placenta, as indicated by virtual northern blot analysis. Inaddition, PCR-based tissue screening panels indicate expression in humanleukocytes. Modulation includes both up-regulation (i.e. activation oragonization) or down-regulation (suppression or antagonization) ofnucleic acid expression.

[0141] Alternatively, a modulator for Ras-like protein nucleic acidexpression can be a small molecule or drug identified using thescreening assays described herein as long as the drug or small moleculeinhibits the Ras-like protein nucleic acid expression in the cells andtissues that express the protein. Experimental data as provided in FIG.1 indicates expression in humans in B-cells from tonsils, adrenal cortexcarcinomas, Jurkat T-cells, prostate adenocarcinomas, lung (includingnormal and small cell carcinoma), marrow, placenta, and leukocytes.

[0142] The nucleic acid molecules are also useful for monitoring theeffectiveness of modulating compounds on the expression or activity ofthe Ras-like protein gene in clinical trials or in a treatment regimen.Thus, the gene expression pattern can serve as a barometer for thecontinuing effectiveness of treatment with the compound, particularlywith compounds to which a patient can develop resistance. The geneexpression pattern can also serve as a marker indicative of aphysiological response of the affected cells to the compound.Accordingly, such monitoring would allow either increased administrationof the compound or the administration of alternative compounds to whichthe patient has not become resistant. Similarly, if the level of nucleicacid expression falls below a desirable level, administration of thecompound could be commensurately decreased.

[0143] The nucleic acid molecules are also useful in diagnostic assaysfor qualitative changes in Ras-like protein nucleic acid, andparticularly in qualitative changes that lead to pathology. The nucleicacid molecules can be used to detect mutations in Ras-like protein genesand gene expression products such as mRNA. The nucleic acid moleculescan be used as hybridization probes to detect naturally occurringgenetic mutations in the Ras-like protein gene and thereby to determinewhether a subject with the mutation is at risk for a disorder caused bythe mutation. Mutations include deletion, addition, or substitution ofone or more nucleotides in the gene, chromosomal rearrangement, such asinversion or transposition, modification of genomic DNA, such asaberrant methylation patterns, or changes in gene copy number, such asamplification. Detection of a mutated form of the Ras-like protein geneassociated with a dysfunction provides a diagnostic tool for an activedisease or susceptibility to disease when the disease results fromoverexpression, underexpression, or altered expression of a Ras-likeprotein.

[0144] Individuals carrying mutations in the Ras-like protein gene canbe detected at the nucleic acid level by a variety of techniques. Thegene encoding the novel Ras-like protein of the present invention islocated on a genome component that has been mapped to human chromosome 9(as indicated in FIG. 3), which is supported by multiple lines ofevidence, such as STS and BAC map data. Genomic DNA can be analyzeddirectly or can be amplified by using PCR prior to analysis. RNA or cDNAcan be used in the same way. In some uses, detection of the mutationinvolves the use of a probe/primer in a polymerase chain reaction (PCR)(see, e.g. U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCRor RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see,e.g., Landegran et al., Science 241:1077-1080 (1988); and Nakazawa etal., PNAS 91:360-364 (1994)), the latter of which can be particularlyuseful for detecting point mutations in the gene (see Abravaya et al.,Nucleic Acids Res. 23:675-682 (1995)). This method can include the stepsof collecting a sample of cells from a patient, isolating nucleic acid(e.g., genomic, mRNA or both) from the cells of the sample, contactingthe nucleic acid sample with one or more primers which specificallyhybridize to a gene under conditions such that hybridization andamplification of the gene (if present) occurs, and detecting thepresence or absence of an amplification product, or detecting the sizeof the amplification product and comparing the length to a controlsample. Deletions and insertions can be detected by a change in size ofthe amplified product compared to the normal genotype. Point mutationscan be identified by hybridizing amplified DNA to normal RNA orantisense DNA sequences.

[0145] Alternatively, mutations in a Ras-like protein gene can bedirectly identified, for example, by alterations in restriction enzymedigestion patterns determined by gel electrophoresis.

[0146] Further, sequence-specific ribozymes (U.S. Pat. No. 5,498,531)can be used to score for the presence of specific mutations bydevelopment or loss of a ribozyme cleavage site. Perfectly matchedsequences can be distinguished from mismatched sequences by nucleasecleavage digestion assays or by differences in melting temperature.

[0147] Sequence changes at specific locations can also be assessed bynuclease protection assays such as RNase and S1 protection or thechemical cleavage method. Furthermore, sequence differences between amutant Ras-like protein gene and a wild-type gene can be determined bydirect DNA sequencing. A variety of automated sequencing procedures canbe utilized when performing the diagnostic assays (Naeve, C. W.,Biotechniques 19:448 (1995)), including sequencing by mass spectrometry(see, e.g., PCT International Publication No. WO 94/16101; Cohen et al.,Adv. Chromatogr. 36:127-162 (1996); and Griffin et al., Appl. Biochem.Biotechnol. 38:147-159 (1993)).

[0148] Other methods for detecting mutations in the gene include methodsin which protection from cleavage agents is used to detect mismatchedbases in RNA/RNA or RNA/DNA duplexes (Myers et al., Science 230:1242(1985)); Cotton et al., PNAS 85:4397 (1988); Saleeba et al., Meth.Enzymol. 217:286-295 (1992)), electrophoretic mobility of mutant andwild type nucleic acid is compared (Orita et al., PNAS 86:2766 (1989);Cotton et al., Mutat. Res. 285:125-144 (1993); and Hayashi et al.,Genet. Anal. Tech. AppL. 9:73-79 (1992)), and movement of mutant orwild-type fragments in polyacrylamide gels containing a gradient ofdenaturant is assayed using denaturing gradient gel electrophoresis(Myers et al., Nature 313:495 (1985)). Examples of other techniques fordetecting point mutations include, selective oligonucleotidehybridization, selective amplification, and selective primer extension.

[0149] The nucleic acid molecules are also useful for testing anindividual for a genotype that while not necessarily causing thedisease, nevertheless affects the treatment modality. Thus, the nucleicacid molecules can be used to study the relationship between anindividual's genotype and the individual's response to a compound usedfor treatment (pharmacogenomic relationship). Accordingly, the nucleicacid molecules described herein can be used to assess the mutationcontent of the Ras-like protein gene in an individual in order to selectan appropriate compound or dosage regimen for treatment.

[0150] Thus nucleic acid molecules displaying genetic variations thataffect treatment provide a diagnostic target that can be used to tailortreatment in an individual. Accordingly, the production of recombinantcells and animals containing these polymorphisms allow effectiveclinical design of treatment compounds and dosage regimens.

[0151] The nucleic acid molecules are thus useful as antisenseconstructs to control Ras-like protein gene expression in cells,tissues, and organisms. A DNA antisense nucleic acid molecule isdesigned to be complementary to a region of the gene involved intranscription, preventing transcription and hence production of Ras-likeprotein. An antisense RNA or DNA nucleic acid molecule would hybridizeto the mRNA and thus block translation of mRNA into Ras-like protein.

[0152] Alternatively, a class of antisense molecules can be used toinactivate mRNA in order to decrease expression of Ras-like proteinnucleic acid. Accordingly, these molecules can treat a disordercharacterized by abnormal or undesired Ras-like protein nucleic acidexpression. This technique involves cleavage by means of ribozymescontaining nucleotide sequences complementary to one or more regions inthe mRNA that attenuate the ability of the mRNA to be translated.Possible regions include coding regions and particularly coding regionscorresponding to the catalytic and other functional activities of theRas-like protein, such as ligand binding.

[0153] The nucleic acid molecules also provide vectors for gene therapyin patients containing cells that are aberrant in Ras-like protein geneexpression. Thus, recombinant cells, which include the patient's cellsthat have been engineered ex vivo and returned to the patient, areintroduced into an individual where the cells produce the desiredRas-like protein to treat the individual.

[0154] The invention also encompasses kits for detecting the presence ofa Ras-like protein nucleic acid in a biological sample. Experimentaldata as provided in FIG. 1 indicates that Ras-like proteins of thepresent invention are expressed in humans in B-cells from tonsils,adrenal cortex carcinomas, Jurkat T-cells, prostate adenocarcinomas,lung (including normal and small cell carcinoma), marrow, and placenta,as indicated by virtual northern blot analysis. In addition, PCR-basedtissue screening panels indicate expression in human leukocytes. Forexample, the kit can comprise reagents such as a labeled or labelablenucleic acid or agent capable of detecting Ras-like protein nucleic acidin a biological sample; means for determining the amount of Ras-likeprotein nucleic acid in the sample; and means for comparing the amountof Ras-like protein nucleic acid in the sample with a standard. Thecompound or agent can be packaged in a suitable container. The kit canfurther comprise instructions for using the kit to detect Ras-likeprotein mRNA or DNA.

Nucleic Acid Arrays

[0155] The present invention further provides arrays or microarrays ofnucleic acid molecules that are based on the sequence informationprovided in FIGS. 1 and 3 (SEQ ID NOS:1 and 3).

[0156] As used herein “Arrays” or “Microarrays” refers to an array ofdistinct polynucleotides or oligonucleotides synthesized on a substrate,such as paper, nylon or other type of membrane, filter, chip, glassslide, or any other suitable solid support. In one embodiment, themicroarray is prepared and used according to the methods described inU.S. Pat. No. 5,837,832, Chee et al., PCT application WO95/11995 (Cheeet al.), Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) andSchena, M. et al. (1996; Proc. Natl. Acad. Sci. 93: 10614-10619), all ofwhich are incorporated herein in their entirety by reference. In otherembodiments, such arrays are produced by the methods described by Brownet. al., U.S. Pat. No. 5,807,522.

[0157] The microarray is preferably composed of a large number ofunique, single-stranded nucleic acid sequences, usually either syntheticantisense oligonucleotides or fragments of cDNAs, fixed to a solidsupport. The oligonucleotides are preferably about 6-60 nucleotides inlength, more preferably 15-30 nucleotides in length, and most preferablyabout 20-25 nucleotides in length. For a certain type of microarray, itmay be preferable to use oligonucleotides that are only 7-20 nucleotidesin length. The microarray may contain oligonucleotides that cover theknown 5′, or 3′, sequence, sequential oligonucleotides that cover thefull-length sequence; or unique oligonucleotides selected fromparticular areas along the length of the sequence. Polynucleotides usedin the microarray may be oligonucleotides that are specific to a gene orgenes of interest.

[0158] In order to produce oligonucleotides to a known sequence for amicroarray, the gene(s) of interest (or an ORF identified from thecontigs of the present invention) is typically examined using a computeralgorithm that starts at the 5′ or at the 3′ end of the nucleotidesequence. Typical algorithms will then identify oligomers of definedlength that are unique to the gene, have a GC content within a rangesuitable for hybridization, and lack predicted secondary structure thatmay interfere with hybridization. In certain situations it may beappropriate to use pairs of oligonucleotides on a microarray. The“pairs” will be identical, except for one nucleotide that preferably islocated in the center of the sequence. The second oligonucleotide in thepair (mismatched by one) serves as a control. The number ofoligonucleotide pairs may range from two to one million. The oligomersare synthesized at designated areas on a substrate using alight-directed chemical process. The substrate may be paper, nylon orother type of membrane, filter, chip, glass slide or any other suitablesolid support.

[0159] In another aspect, an oligonucleotide may be synthesized on thesurface of the substrate by using a chemical coupling procedure and anink jet application apparatus, as described in PCT application W095/251116 (Baldeschweiler et al.) which is incorporated herein in itsentirety by reference. In another aspect, a “gridded” array analogous toa dot (or slot) blot may be used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system,thermal, UV, mechanical or chemical bonding procedures. An array, suchas those described above, may be produced by hand or by using availabledevices (slot blot or dot blot apparatus), materials (any suitable solidsupport), and machines (including robotic instruments), and may contain8, 24, 96, 384, 1536, 6144 or more oligonucleotides, or any other numberbetween two and one million which lends itself to the efficient use ofcommercially available instrumentation.

[0160] In order to conduct sample analysis using a microarray, the RNAor DNA from a biological sample is made into hybridization probes. ThemRNA is isolated, and cDNA is produced and used as a template to makeantisense RNA (aRNA). The aRNA is amplified in the presence offluorescent nucleotides, and labeled probes are incubated with themicroarray so that the probe sequences hybridize to complementaryoligonucleotides of the microarray. Incubation conditions are adjustedso that hybridization occurs with precise complementary matches or withvarious degrees of less complementarity. After removal of nonhybridizedprobes, a scanner is used to determine the levels and patterns offluorescence. The scanned images are examined to determine degree ofcomplementarity and the relative abundance of each oligonucleotidesequence on the microarray. The biological samples may be obtained fromany bodily fluids (such as blood, urine, saliva, phlegm, gastric juices,etc.), cultured cells, biopsies, or other tissue preparations. Adetection system may be used to measure the absence, presence, andamount of hybridization for all of the distinct sequencessimultaneously. This data may be used for large-scale correlationstudies on the sequences, expression patterns, mutations, variants, orpolymorphisms among samples.

[0161] Using such arrays, the present invention provides methods toidentify the expression of one or more of the proteins/peptides of thepresent invention. In detail, such methods comprise incubating a testsample with one or more nucleic acid molecules and assaying for bindingof the nucleic acid molecule with components within the test sample.Such assays will typically involve arrays comprising many genes, atleast one of which is a gene of the present invention.

[0162] Conditions for incubating a nucleic acid molecule with a testsample vary. Incubation conditions depend on the format employed in theassay, the detection methods employed, and the type and nature of thenucleic acid molecule used in the assay. One skilled in the art willrecognize that any one of the commonly available hybridization,amplification or array assay formats can readily be adapted to employthe novel fragments of the human genome disclosed herein. Examples ofsuch assays can be found in Chard, T, An Introduction toRadioimmunoassay and Related Techniques, Elsevier Science Publishers,Amsterdam, The Netherlands (1986); Bullock, G. R. et al., Techniques inImmunocytochemistry, Academic Press, Orlando, Fla. Vol. 1 (1 982), Vol.2 (1983), Vol. 3 (1985); Tijssen, P., Practice and Theory of enzymeImmunoassays: Laboratory Techniques in Biochemistry and MolecularBiology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985).

[0163] The test samples of the present invention include cells, proteinor membrane extracts of cells. The test sample used in theabove-described method will vary based on the assay format, nature ofthe detection method and the tissues, cells or extracts used as thesample to be assayed. Methods for preparing nucleic acid extracts or ofcells are well known in the art and can be readily be adapted in orderto obtain a sample that is compatible with the system utilized.

[0164] In another embodiment of the present invention, kits are providedwhich contain the necessary reagents to carry out the assays of thepresent invention.

[0165] Specifically, the invention provides a compartmentalized kit toreceive, in close confinement, one or more containers which comprises:(a) a first container comprising one of the nucleic acid molecules thatcan bind to a fragment of the human genome disclosed herein; and (b) oneor more other containers comprising one or more of the following: washreagents, reagents capable of detecting presence of a bound nucleicacid. Preferred kits will include chips that are capable of detectingthe expression of 10 or more, 100 or more, or 500 or more, 1000 or more,or all of the genes expressed in Human.

[0166] In detail, a compartmentalized kit includes any kit in whichreagents are contained in separate containers. Such containers includesmall glass containers, plastic containers, strips of plastic, glass orpaper, or arraying material such as silica. Such containers allows oneto efficiently transfer reagents from one compartment to anothercompartment such that the samples and reagents are notcross-contaminated, and the agents or solutions of each container can beadded in a quantitative fashion from one compartment to another. Suchcontainers will include a container which will accept the test sample, acontainer which contains the nucleic acid probe, containers whichcontain wash reagents (such as phosphate buffered saline, Tris-buffers,etc.), and containers which contain the reagents used to detect thebound probe. One skilled in the art will readily recognize that thepreviously unidentified Ras-like protein genes of the present inventioncan be routinely identified using the sequence information disclosedherein can be readily incorporated into one of the established kitformats which are well known in the art, particularly expression arrays.

Vectors/Host Cells

[0167] The invention also provides vectors containing the nucleic acidmolecules described herein. The term “vector” refers to a vehicle,preferably a nucleic acid molecule, which can transport the nucleic acidmolecules. When the vector is a nucleic acid molecule, the nucleic acidmolecules are covalently linked to the vector nucleic acid. With thisaspect of the invention, the vector includes a plasmid, single or doublestranded phage, a single or double stranded RNA or DNA viral vector, orartificial chromosome, such as a BAC, PAC, YAC, OR MAC.

[0168] A vector can be maintained in the host cell as anextrachromosomal element where it replicates and produces additionalcopies of the nucleic acid molecules. Alternatively, the vector mayintegrate into the host cell genome and produce additional copies of thenucleic acid molecules when the host cell replicates.

[0169] The invention provides vectors for the maintenance (cloningvectors) or vectors for expression (expression vectors) of the nucleicacid molecules. The vectors can function in procaryotic or eukaryoticcells or in both (shuttle vectors).

[0170] Expression vectors contain cis-acting regulatory regions that areoperably linked in the vector to the nucleic acid molecules such thattranscription of the nucleic acid molecules is allowed in a host cell.The nucleic acid molecules can be introduced into the host cell with aseparate nucleic acid molecule capable of affecting transcription. Thus,the second nucleic acid molecule may provide a trans-acting factorinteracting with the cis-regulatory control region to allowtranscription of the nucleic acid molecules from the vector.Alternatively, a trans-acting factor may be supplied by the host cell.Finally, a trans-acting factor can be produced from the vector itself.It is understood, however, that in some embodiments, transcriptionand/or translation of the nucleic acid molecules can occur in acell-free system.

[0171] The regulatory sequence to which the nucleic acid moleculesdescribed herein can be operably linked include promoters for directingmRNA transcription. These include, but are not limited to, the leftpromoter from bacteriophage λ, the lac, TRP, and TAC promoters from E.coli, the early and late promoters from SV40, the CMV immediate earlypromoter, the adenovirus early and late promoters, and retroviruslong-terminal repeats.

[0172] In addition to control regions that promote transcription,expression vectors may also include regions that modulate transcription,such as repressor binding sites and enhancers. Examples include the SV40enhancer, the cytomegalovirus immediate early enhancer, polyomaenhancer, adenovirus enhancers, and retrovirus LTR enhancers.

[0173] In addition to containing sites for transcription initiation andcontrol, expression vectors can also contain sequences necessary fortranscription termination and, in the transcribed region a ribosomebinding site for translation. Other regulatory control elements forexpression include initiation and termination codons as well aspolyadenylation signals. The person of ordinary skill in the art wouldbe aware of the numerous regulatory sequences that are useful inexpression vectors. Such regulatory sequences are described, forexample, in Sambrook et al., Molecular Cloning: A Laboratory Manual.2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,(1989).

[0174] A variety of expression vectors can be used to express a nucleicacid molecule. Such vectors include chromosomal, episomal, andvirus-derived vectors, for example vectors derived from bacterialplasmids, from bacteriophage, from yeast episomes, from yeastchromosomal elements, including yeast artificial chromosomes, fromviruses such as baculoviruses, papovaviruses such as SV40, Vacciniaviruses, adenoviruses, poxviruses, pseudorabies viruses, andretroviruses. Vectors may also be derived from combinations of thesesources such as those derived from plasmid and bacteriophage geneticelements, e.g. cosmids and phagemids. Appropriate cloning and expressionvectors for prokaryotic and eukaryotic hosts are described in Sambrooket al., Molecular Cloning: A Laboratory Manual. 2nd. ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., (1989).

[0175] The regulatory sequence may provide constitutive expression inone or more host cells (i.e. tissue specific) or may provide forinducible expression in one or more cell types such as by temperature,nutrient additive, or exogenous factor such as a hormone or otherligand. A variety of vectors providing for constitutive and inducibleexpression in prokaryotic and eukaryotic hosts are well known to thoseof ordinary skill in the art.

[0176] The nucleic acid molecules can be inserted into the vectornucleic acid by well-known methodology. Generally, the DNA sequence thatwill ultimately be expressed is joined to an expression vector bycleaving the DNA sequence and the expression vector with one or morerestriction enzymes and then ligating the fragments together. Proceduresfor restriction enzyme digestion and ligation are well known to those ofordinary skill in the art.

[0177] The vector containing the appropriate nucleic acid molecule canbe introduced into an appropriate host cell for propagation orexpression using well-known techniques.

[0178] Bacterial cells include, but are not limited to, E. coli,Streptomyces, and Salmonella typhimurium. Eukaryotic cells include, butare not limited to, yeast, insect cells such as Drosophila, animal cellssuch as COS and CHO cells, and plant cells.

[0179] As described herein, it may be desirable to express the peptideas a fusion protein.

[0180] Accordingly, the invention provides fusion vectors that allow forthe production of the peptides. Fusion vectors can increase theexpression of a recombinant protein, increase the solubility of therecombinant protein, and aid in the purification of the protein byacting for example as a ligand for affinity purification. A proteolyticcleavage site may be introduced at the junction of the fusion moiety sothat the desired peptide can ultimately be separated from the fusionmoiety. Proteolytic enzymes include, but are not limited to, factor Xa,thrombin, and enteroRas-like protein. Typical fusion expression vectorsinclude pGEX (Smith et al., Gene 67:31-40 (1988)), pMAL (New EnglandBiolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) whichfuse glutathione S-transferase (GST), maltose E binding protein, orprotein A, respectively, to the target recombinant protein. Examples ofsuitable inducible non-fusion E. coli expression vectors include pTrc(Amann et al., Gene 69:301-315 (1988)) and pET 11d (Studier et al., GeneExpression Technology: Methods in Enzymology 185:60-89 (1990)).

[0181] Recombinant protein expression can be maximized in a hostbacteria by providing a genetic background wherein the host cell has animpaired capacity to proteolytically cleave the recombinant protein.(Gottesman, S., Gene Expression Technology: Methods in Enzymology 185,Academic Press, San Diego, Calif. (1990) 119-128). Alternatively, thesequence of the nucleic acid molecule of interest can be altered toprovide preferential codon usage for a specific host cell, for exampleE. coli. (Wada et al., Nucleic Acids Res. 20:2111-2118 (1992)).

[0182] The nucleic acid molecules can also be expressed by expressionvectors that are operative in yeast. Examples of vectors for expressionin yeast e.g., S. cerevisiae include pYepSec1 (Baldari, et al., EMBO J6:229-234 (1987)), pMFa (Kurjan et al., Cell 30:933-943(1982)), pJRY88(Schultz et al., Gene 54:113-123 (1987)), and pYES2 (InvitrogenCorporation, San Diego, Calif.).

[0183] The nucleic acid molecules can also be expressed in insect cellsusing, for example, baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., Sf9cells) include the pAc series (Smith et al., Mol. Cell Biol. 3:2156-2165(1983)) and the pVL series (Lucklow et al., Virology 170:31-39 (1989)).

[0184] In certain embodiments of the invention, the nucleic acidmolecules described herein are expressed in mammalian cells usingmammalian expression vectors. Examples of mammalian expression vectorsinclude pCDM8 (Seed, B. Nature 329:840(1987)) and pMT2PC (Kaufman etal., EMBO J. 6:187-195 (1987)).

[0185] The expression vectors listed herein are provided by way ofexample only of the well-known vectors available to those of ordinaryskill in the art that would be useful to express the nucleic acidmolecules. The person of ordinary skill in the art would be aware ofother vectors suitable for maintenance, propagation, or expression ofthe nucleic acid molecules described herein. These are found for examplein Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: ALaboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

[0186] The invention also encompasses vectors in which the nucleic acidsequences described herein are cloned into the vector in reverseorientation, but operably linked to a regulatory sequence that permitstranscription of antisense RNA. Thus, an antisense transcript can beproduced to all, or to a portion, of the nucleic acid molecule sequencesdescribed herein, including both coding and non-coding regions.Expression of this antisense RNA is subject to each of the parametersdescribed above in relation to expression of the sense RNA (regulatorysequences, constitutive or inducible expression, tissue-specificexpression).

[0187] The invention also relates to recombinant host cells containingthe vectors described herein. Host cells therefore include prokaryoticcells, lower eukaryotic cells such as yeast, other eukaryotic cells suchas insect cells, and higher eukaryotic cells such as mammalian cells.

[0188] The recombinant host cells are prepared by introducing the vectorconstructs described herein into the cells by techniques readilyavailable to the person of ordinary skill in the art. These include, butare not limited to, calcium phosphate transfection,DEAE-dextran-mediated transfection, cationic lipid-mediatedtransfection, electroporation, transduction, infection, lipofection, andother techniques such as those found in Sambrook, et al. (MolecularCloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

[0189] Host cells can contain more than one vector. Thus, differentnucleotide sequences can be introduced on different vectors of the samecell. Similarly, the nucleic acid molecules can be introduced eitheralone or with other nucleic acid molecules that are not related to thenucleic acid molecules such as those providing trans-acting factors forexpression vectors. When more than one vector is introduced into a cell,the vectors can be introduced independently, co-introduced, or joined tothe nucleic acid molecule vector.

[0190] In the case of bacteriophage and viral vectors, these can beintroduced into cells as packaged or encapsulated virus by standardprocedures for infection and transduction. Viral vectors can bereplication-competent or replication-defective. In the case in whichviral replication is defective, replication will occur in host cellsproviding functions that complement the defects.

[0191] Vectors generally include selectable markers that enable theselection of the subpopulation of cells that contain the recombinantvector constructs. The marker can be contained in the same vector thatcontains the nucleic acid molecules described herein or may be on aseparate vector. Markers include tetracycline or ampicillin-resistancegenes for prokaryotic host cells and dihydrofolate reductase or neomycinresistance for eukaryotic host cells. However, any marker that providesselection for a phenotypic trait will be effective.

[0192] While the mature proteins can be produced in bacteria, yeast,mammalian cells, and other cells under the control of the appropriateregulatory sequences, cell-free transcription and translation systemscan also be used to produce these proteins using RNA derived from theDNA constructs described herein.

[0193] Where secretion of the peptide is desired, which is difficult toachieve with multi-transmembrane domain containing proteins such askinases, appropriate secretion signals are incorporated into the vector.The signal sequence can be endogenous to the peptides or heterologous tothese peptides.

[0194] Where the peptide is not secreted into the medium, which istypically the case with kinases, the protein can be isolated from thehost cell by standard disruption procedures, including freeze thaw,sonication, mechanical disruption, use of lysing agents and the like.The peptide can then be recovered and purified by well-knownpurification methods including ammonium sulfate precipitation, acidextraction, anion or cationic exchange chromatography, phosphocellulosechromatography, hydrophobic-interaction chromatography, affinitychromatography, hydroxylapatite chromatography, lectin chromatography,or high performance liquid chromatography.

[0195] It is also understood that depending upon the host cell inrecombinant production of the peptides described herein, the peptidescan have various glycosylation patterns, depending upon the cell, ormaybe non-glycosylated as when produced in bacteria. In addition, thepeptides may include an initial modified methionine in some cases as aresult of a host-mediated process.

Uses of Vectors and Host Cells

[0196] The recombinant host cells expressing the peptides describedherein have a variety of uses. First, the cells are useful for producinga Ras-like protein polypeptide that can be further purified to producedesired amounts of Ras-like protein or fragments. Thus, host cellscontaining expression vectors are useful for peptide production.

[0197] Host cells are also useful for conducting cell-based assaysinvolving the Ras-like protein or Ras-like protein fragments. Thus, arecombinant host cell expressing a native Ras-like protein is useful forassaying compounds that stimulate or inhibit Ras-like protein function.

[0198] Host cells are also useful for identifying Ras-like proteinmutants in which these functions are affected. If the mutants naturallyoccur and give rise to a pathology, host cells containing the mutationsare useful to assay compounds that have a desired effect on the mutantRas-like protein (for example, stimulating or inhibiting function) whichmay not be indicated by their effect on the native Ras-like protein.

[0199] Genetically engineered host cells can be further used to producenon-human transgenic animals. A transgenic animal is preferably amammal, for example a rodent, such as a rat or mouse, in which one ormore of the cells of the animal include a transgene. A transgene isexogenous DNA which is integrated into the genome of a cell from which atransgenic animal develops and which remains in the genome of the matureanimal in one or more cell types or tissues of the transgenic animal.These animals are useful for studying the function of a Ras-like proteinand identifying and evaluating modulators of Ras-like protein activity.Other examples of transgenic animals include non-human primates, sheep,dogs, cows, goats, chickens, and amphibians.

[0200] A transgenic animal can be produced by introducing nucleic acidinto the male pronuclei of a fertilized oocyte, e.g., by microinjection,retroviral infection, and allowing the oocyte to develop in apseudopregnant female foster animal. Any of the Ras-like proteinnucleotide sequences can be introduced as a transgene into the genome ofa non-human animal, such as a mouse.

[0201] Any of the regulatory or other sequences useful in expressionvectors can form part of the transgenic sequence. This includes intronicsequences and polyadenylation signals, if not already included. Atissue-specific regulatory sequence(s) can be operably linked to thetransgene to direct expression of the Ras-like protein to particularcells.

[0202] Methods for generating transgenic animals via embryo manipulationand microinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No.4,873,191 by Wagner et al. and in Hogan, B., Manipulating the MouseEmbryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986). Similar methods are used for production of other transgenicanimals. A transgenic founder animal can be identified based upon thepresence of the transgene in its genome and/or expression of transgenicmRNA in tissues or cells of the animals. A transgenic founder animal canthen be used to breed additional animals carrying the transgene.Moreover, transgenic animals carrying a transgene can further be bred toother transgenic animals carrying other transgenes. A transgenic animalalso includes animals in which the entire animal or tissues in theanimal have been produced using the homologously recombinant host cellsdescribed herein.

[0203] In another embodiment, transgenic non-human animals can beproduced which contain selected systems which allow for regulatedexpression of the transgene. One example of such a system is thecre/loxP recombinase system of bacteriophage P1. For a description ofthe cre/loxP recombinase system, see, e.g., Lakso et al. PNAS89:6232-6236 (1992). Another example of a recombinase system is the FLPrecombinase system of S. cerevisiae (O'Gorman et al. Science251:1351-1355 (1991). If a cre/loxP recombinase system is used toregulate expression of the transgene, animals containing transgenesencoding both the Cre recombinase and a selected protein is required.Such animals can be provided through the construction of “double”transgenic animals, e.g., by mating two transgenic animals, onecontaining a transgene encoding a selected protein and the othercontaining a transgene encoding a recombinase.

[0204] Clones of the non-human transgenic animals described herein canalso be produced according to the methods described in Wilmut, I. et al.Nature 385:810-813 (1997) and PCT International Publication Nos. WO97/07668 and WO 97/07669. In brief, a cell, e.g., a somatic cell, fromthe transgenic animal can be isolated and induced to exit the growthcycle and enter G_(o) phase. The quiescent cell can then be fused, e.g.,through the use of electrical pulses, to an enucleated oocyte from ananimal of the same species from which the quiescent cell is isolated.The reconstructed oocyte is then cultured such that it develops tomorula or blastocyst and then transferred to pseudopregnant femalefoster animal. The offspring born of this female foster animal will be aclone of the animal from which the cell, e.g., the somatic cell, isisolated.

[0205] Transgenic animals containing recombinant cells that express thepeptides described herein are useful to conduct the assays describedherein in an in vivo context. Accordingly, the various physiologicalfactors that are present in vivo and that could effect ligand binding,Ras-like protein activation, and signal transduction, may not be evidentfrom in vitro cell-free or cell-based assays. Accordingly, it is usefulto provide non-human transgenic animals to assay in vivo Ras-likeprotein function, including ligand interaction, the effect of specificmutant Ras-like proteins on Ras-like protein function and ligandinteraction, and the effect of chimeric Ras-like proteins. It is alsopossible to assess the effect of null mutations, which is mutations thatsubstantially or completely eliminate one or more Ras-like proteinfunctions.

[0206] All publications and patents mentioned in the above specificationare herein incorporated by reference. Various modifications andvariations of the described method and system of the invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of theabove-described modes for carrying out the invention, which are obviousto those skilled in the field of molecular biology or related fields,are intended to be within the scope of the following claims.

1 5 1 3380 DNA Human 1 ggaggatggt ggttgtgggt cctctgtgag cagccctgttaacctttaaa ccagcacggc 60 cgccgcagct gtgagcctag cacctgatca gtggagagctgtggattgca tgtttgtttg 120 ccattgcccc cgccaccctg caagttgcac cttctagaatcagcaagcca agctcctctc 180 acccagcgta atgatgcgga aatgcaaatg caccatcatgttgtgaccca tattgcgaaa 240 attagaaaaa aggaagttgt gtttcgctat tgcacgaagttcagcccaga ggagaaactc 300 gctcgccttc agaagacagt acctcctaaa tggctctactttgaacctgc tgggcaagga 360 agagattttc aaggaaacca tctaccgtgt gcaagctcctgccggccaac cccagacccc 420 agcacggagc caggcgcctg tgcccgccga ccctcagcatcctcctcaga aaggctggtg 480 gcatcaggaa gcccctggcc agcctccacc tgagcccagtgagctcagct ttaaggatgg 540 agtcaggcag ggggtcctca acccctccag gacccattgctgccctaggg atgccagaca 600 ctgggcctgg cagttcctcc ctagggaagc ttcaggcgctccctgttggg cccagagccc 660 actgtgggga ccctgtcagc ctggctgcag caggggacggctctccagac ataggcccca 720 cgggagagct gagtggtagc ttaaagatcc ccaaccgggacagcgggatc gacagtccct 780 cctccagtgt ggctggagag aactttccct gcgaggagggcttggaggct ggcccaagcc 840 ccactgtact gggggcgcac gcagagatgg ccctggacagccaggtcccg aaggtcaccc 900 cccaggagga ggcggacagc gacgtgggtg aggaacctgactctgagaac accccccaga 960 aggctgacaa ggatgccggc ctggcccagc actctggcccccagaagctt ctccacattg 1020 cccaggagct cctgcacacc gaggagacct atgtgaagcggctgcacctg ctggaccagg 1080 ttttctgcac caggctgacg gatgcgggga tccctccagaagtcatcatg ggcatattct 1140 ctaacatctc ctccatccac cgcttccacg ggcagttcctgctgccggag ctgaagacgc 1200 ggatcacgga ggagtgggac acaaacccac ggctcggggacatcctgcag aagctggccc 1260 cattcctgaa gatgtacggc gagtatgtca agaactttgaccgagccgta gggctggtga 1320 gcacgtggac ccagcgctcc ccactgttta aagacgtcgtccacagcatc cagaagcagg 1380 aggtatgcgg gaacctgacg ctgcagcacc acatgctggagcccgtgcag agggtccccc 1440 ggtacgagct gctgctcaag gactatctga agaggctcccgcaggacgcc ccagaccgga 1500 aggatgcgga gaggtccttg gagctcatct ccacagccgccaaccactcc aatgctgcca 1560 ttcggaaagt ggagaaaatg cacaagctct tggaggtgtacgagcagctg ggtggggaag 1620 aagacattgt caacccggcc aatgaactga tcaaggagggccaaatccag aaactgtcag 1680 ccaagaacgg caccccccag gaccgccacc tcttcctgttcaacagcatg atcctttact 1740 gtgtgcccaa gctgcggctc atgggccaga agttcagcgtccgggagaag atggacatct 1800 caggcctcca ggtgcaggat atcgtcaagc caaacacagcacatacattc atcataacgg 1860 gaagaaaaag gtccctggag ctgcagacgc ggacagaggaagagaagaaa gaatggattc 1920 agatcatcca ggccaccatc gagaagcaca aacagaacagcgaaaccttc aaggcttttg 1980 gtggcgcctt cagccaggat gaggacccca gcctctctccagacatgcct atcacgagca 2040 ccagccctgt ggagcctgtg gtgaccaccg aaggcagttcgggtgcagca gggctcgagc 2100 ccagaaaact atcctctaag accagacgtg acaaggagaagcagagctgt aagagctgtg 2160 gtgagacctt caactccatc accaagagga ggcatcactgcaagctgtgt ggggcggtca 2220 tctgtgggaa gtgctccgag ttcaaggccg agaacagccggcagagccgt gtctgcagag 2280 attgtttcct gacacagcca gtggcccctg agagcacagagaagacaccc actgcagacc 2340 cccagcccag cctgctctgc ggccccctgc ggctgtcagagagcggtgag acctggagcg 2400 aggtgtgggc cgccatcccc atgtcagatc cccaggtgctgcacctgcag ggaggcagcc 2460 aggacggccg gctgccccgc accatccctc tccccagctgcaaactgagt gtgccggacc 2520 ctgaggagag gctggactcg gggcatgtgt ggaagctgcagtgggccaag cagtcctggt 2580 acctgagcgc ctcctccgca gagctgcagc agcagtggctggaaacccta agcactgctg 2640 cccatgggga cacggcccag gacagcccgg gggccctgcagcttcaggtc cctatgggcg 2700 cagctgctcc gtgagctgag tctcccactg ccctgcacaccaccacattg gacctgtgct 2760 gtcctgggag gtggtgttgg aggccccatg aagagcgccctggactgctg agggtgggcc 2820 aacagcccag agctcaggac acttggcttt ggggggaaggaaactgaggc ccagagaggg 2880 gcaaccactg gccaagggtc acccagcaag ttttggctaagagcctggcc tccagcccca 2940 gcagtgtggc ccagagcagg ggccgactgc caaagtaaccatcatccata tgggccgtgt 3000 ggtgatgctg gcccggaagg cagaaagagg cagcatgggcactgccaggg acagccacat 3060 cctgctggtc tgcagcgtgg tccaccccgc ctctgcccagcctgtctaca ccgtgtgagc 3120 tgaatcgtga cttgcttccc acctcctttc tctgtcctctcctgaggttc tgcctgcagc 3180 ccccaggagg tgggcctgcc ccatcctagc tggactcatggttcctaaat aaccacgctc 3240 agaagctctg ctaggactta ccccagccac tgagtggcaggcgcatgaga tttgtggctg 3300 ttcctgatgc tagtggcaca cagtgcttat ctgcataaataaacactggc caccaaaaaa 3360 aaaaaaaaaa aaaaaaaaaa 3380 2 725 PRT Human 2Met Glu Ser Gly Arg Gly Ser Ser Thr Pro Pro Gly Pro Ile Ala Ala 1 5 1015 Leu Gly Met Pro Asp Thr Gly Pro Gly Ser Ser Ser Leu Gly Lys Leu 20 2530 Gln Ala Leu Pro Val Gly Pro Arg Ala His Cys Gly Asp Pro Val Ser 35 4045 Leu Ala Ala Ala Gly Asp Gly Ser Pro Asp Ile Gly Pro Thr Gly Glu 50 5560 Leu Ser Gly Ser Leu Lys Ile Pro Asn Arg Asp Ser Gly Ile Asp Ser 65 7075 80 Pro Ser Ser Ser Val Ala Gly Glu Asn Phe Pro Cys Glu Glu Gly Leu 8590 95 Glu Ala Gly Pro Ser Pro Thr Val Leu Gly Ala His Ala Glu Met Ala100 105 110 Leu Asp Ser Gln Val Pro Lys Val Thr Pro Gln Glu Glu Ala AspSer 115 120 125 Asp Val Gly Glu Glu Pro Asp Ser Glu Asn Thr Pro Gln LysAla Asp 130 135 140 Lys Asp Ala Gly Leu Ala Gln His Ser Gly Pro Gln LysLeu Leu His 145 150 155 160 Ile Ala Gln Glu Leu Leu His Thr Glu Glu ThrTyr Val Lys Arg Leu 165 170 175 His Leu Leu Asp Gln Val Phe Cys Thr ArgLeu Thr Asp Ala Gly Ile 180 185 190 Pro Pro Glu Val Ile Met Gly Ile PheSer Asn Ile Ser Ser Ile His 195 200 205 Arg Phe His Gly Gln Phe Leu LeuPro Glu Leu Lys Thr Arg Ile Thr 210 215 220 Glu Glu Trp Asp Thr Asn ProArg Leu Gly Asp Ile Leu Gln Lys Leu 225 230 235 240 Ala Pro Phe Leu LysMet Tyr Gly Glu Tyr Val Lys Asn Phe Asp Arg 245 250 255 Ala Val Gly LeuVal Ser Thr Trp Thr Gln Arg Ser Pro Leu Phe Lys 260 265 270 Asp Val ValHis Ser Ile Gln Lys Gln Glu Val Cys Gly Asn Leu Thr 275 280 285 Leu GlnHis His Met Leu Glu Pro Val Gln Arg Val Pro Arg Tyr Glu 290 295 300 LeuLeu Leu Lys Asp Tyr Leu Lys Arg Leu Pro Gln Asp Ala Pro Asp 305 310 315320 Arg Lys Asp Ala Glu Arg Ser Leu Glu Leu Ile Ser Thr Ala Ala Asn 325330 335 His Ser Asn Ala Ala Ile Arg Lys Val Glu Lys Met His Lys Leu Leu340 345 350 Glu Val Tyr Glu Gln Leu Gly Gly Glu Glu Asp Ile Val Asn ProAla 355 360 365 Asn Glu Leu Ile Lys Glu Gly Gln Ile Gln Lys Leu Ser AlaLys Asn 370 375 380 Gly Thr Pro Gln Asp Arg His Leu Phe Leu Phe Asn SerMet Ile Leu 385 390 395 400 Tyr Cys Val Pro Lys Leu Arg Leu Met Gly GlnLys Phe Ser Val Arg 405 410 415 Glu Lys Met Asp Ile Ser Gly Leu Gln ValGln Asp Ile Val Lys Pro 420 425 430 Asn Thr Ala His Thr Phe Ile Ile ThrGly Arg Lys Arg Ser Leu Glu 435 440 445 Leu Gln Thr Arg Thr Glu Glu GluLys Lys Glu Trp Ile Gln Ile Ile 450 455 460 Gln Ala Thr Ile Glu Lys HisLys Gln Asn Ser Glu Thr Phe Lys Ala 465 470 475 480 Phe Gly Gly Ala PheSer Gln Asp Glu Asp Pro Ser Leu Ser Pro Asp 485 490 495 Met Pro Ile ThrSer Thr Ser Pro Val Glu Pro Val Val Thr Thr Glu 500 505 510 Gly Ser SerGly Ala Ala Gly Leu Glu Pro Arg Lys Leu Ser Ser Lys 515 520 525 Thr ArgArg Asp Lys Glu Lys Gln Ser Cys Lys Ser Cys Gly Glu Thr 530 535 540 PheAsn Ser Ile Thr Lys Arg Arg His His Cys Lys Leu Cys Gly Ala 545 550 555560 Val Ile Cys Gly Lys Cys Ser Glu Phe Lys Ala Glu Asn Ser Arg Gln 565570 575 Ser Arg Val Cys Arg Asp Cys Phe Leu Thr Gln Pro Val Ala Pro Glu580 585 590 Ser Thr Glu Lys Thr Pro Thr Ala Asp Pro Gln Pro Ser Leu LeuCys 595 600 605 Gly Pro Leu Arg Leu Ser Glu Ser Gly Glu Thr Trp Ser GluVal Trp 610 615 620 Ala Ala Ile Pro Met Ser Asp Pro Gln Val Leu His LeuGln Gly Gly 625 630 635 640 Ser Gln Asp Gly Arg Leu Pro Arg Thr Ile ProLeu Pro Ser Cys Lys 645 650 655 Leu Ser Val Pro Asp Pro Glu Glu Arg LeuAsp Ser Gly His Val Trp 660 665 670 Lys Leu Gln Trp Ala Lys Gln Ser TrpTyr Leu Ser Ala Ser Ser Ala 675 680 685 Glu Leu Gln Gln Gln Trp Leu GluThr Leu Ser Thr Ala Ala His Gly 690 695 700 Asp Thr Ala Gln Asp Ser ProGly Ala Leu Gln Leu Gln Val Pro Met 705 710 715 720 Gly Ala Ala Ala Pro725 3 88191 DNA Human misc_feature (1)...(88191) n = A,T,C or G 3ggagcagtgt ggaaggaaat ctggcctcat cccgtggaac cagtgcctca gtttctccat 60ttgtaaatgg ggctatgtgt cacccactgg tggcttgctg ttgggtgaaa gagctgagga 120ctgcattttt gctgtgtgtg acctatacgg aagcctgtca tccatgacag tgtctgacaa 180gcgcctgcca gtctgcagcg gggcgtccgt ccactctggc tatggggaca tgtgggccca 240tttcctcatc atccctgcgt gtcccttccc aagctcatga gcaacagtgc tgactcagtc 300ctgcagcatc agttcagtct tggggtgtcg tcattctcca ggacatccca acgagggata 360tccagcagcc ttccttgagg gtcccaccct cagggacagg atgggtttgg gtggtagcca 420tagctccaca gtctcaggtt gcccagggcc accaggcagc ggctcttggc cctgagtgta 480tctgtgtggt ccaagaagag gcgaggtgct tgtgtgtgca gaaatgtgcc aggaaggaca 540ggggagactc aggcttcctc cacaacaccc ctgttcgagt gtgccagtga ggggtggggc 600tcccttctcc caccacaacc ctctgaggag ctctccctgt cctgctggcc aacgagccct 660gtgcctaccg gcagaggctc aactaaccaa ccaactaacc aacagctgac caactaacca 720accagctaat tcatcaactc cctttctctt ctctgttttc tccttttcct cctactccct 780ccttttcctc ccttccttcc cccctccctt ctttccttcc tctttttttt ttctttttct 840cctcccttca tctgttcacc tgttattcca ggaaggatct ggttcagcat agagtgcaaa 900ggataaaaag aagcagggtg ggggccaggc acagtggctt acgcctgtaa tcccagcact 960ttggggaggc tgaggcaggt ggatcacgag gtcagnnnnn nnnnnnnnnn nnnnnnnnnn 1020nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1080nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1140nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1200nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1260nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1320nnnnnnnnnn nnnnnnnnnn nnnnnacttg gaccaaaact cctgggtccc tgactgatgg 1380tggggaaaga agggggcagg aggggttcat tcaaggctgg caaatgtagc ccgagtccct 1440tctgacccct ggtagggccc tcatgaactg tggaggctgt ggtagtctgt ggctagggtg 1500cctggtgaca tttggtctaa agaattaaat ccagtgcaga tgagtccacc caggaggaat 1560ctgtggacga cagcccctaa gtgacctggg gacggctcag ggacagtgct gccctttctg 1620cctaggcaga tggaaatgga gccagggtcc ttgttgtgcc acgtcccctg cgtccagcac 1680tggagattgg cttctcccca agatctggct gcccctggcc tgcttgggcc ctgaagagaa 1740tgaggagagg caagggtgcc taggagaaag gtggtagagg tggtgggagc cctgcaggct 1800gagggttgtg gccaggggct gctgagacac caggttgctc tgggggtccc gctctaggtc 1860tagtgtgttc atgagggaat tccaaccggc ctttaaggaa tgtggggctt ttagaaggaa 1920aatggtatat agcatactaa tatgtgcttg ttgaatgaat acatgaagaa ggccttttat 1980ttttctagtt actgatatcc agtaattcta agtattccta gcttgtcttc tctgggtaaa 2040ttactttgct tctcagaata tgccagctag acacaaccca taaggtagaa tttttcattg 2100aaatgtgtct tacgtggaac ttgtaacttc ctaaaataaa gttgtgtagc caaacaggat 2160ggctcatgcc tataatccca acactttgag aggccgagat gggtggatgg cttgagtcca 2220ggagttcaag accaggctgg gcaacattgt gaaatccttg tctgcaaaaa atacacaaat 2280aagccaggtt gtggtggcat gcgcctgtag tcccagctac tcgggagact gaggtgggag 2340gattgcttga acccagaggt tgaggctgca gtgagctgtg attgtgccac tgcactctag 2400cctgggtgac agagcgagac cctgtctcaa aaaaattaat taattaatga aagttgtgta 2460gattttgcca gatgtagggt tgtgtctgag cacactcatg ttcttggtcc ttgaaacact 2520ggtgcctgaa ggattcatgt agctgcggag gtggtaaact gaatgaataa aactgctggt 2580ctttgaccat gcaaaatagg tgtaaagtag gcttgggggg atctcagtca gaagaccctg 2640actttacggc tgcagagagg atgcctctgt cccccagctc cagggcagac aaaatctgag 2700ggtctaagat cttcagtggc aaaaggggca tctggttttt tgaccctctc cctgatcttg 2760cttttaaaca agttccaaaa tattttgtct gcaaaacaaa ctatgctgca taaggaataa 2820ttcgtggccc taattcatcc attagagacc acacttgact ccccaagggc ttcttaatat 2880ccctggtttc caactcatgt gagtcaatgc agggggtgac cagagcacat ctctgtttat 2940tttcaagtat gcaaaggata tatacacata tcattgttga aagtcaattt tttttttttt 3000ttttttttgg agatagagtc ttgctctgtc gcccgggctg gagtgcagtg gcacgatctc 3060ggctcactgc aagctccgct tcctggcttc accccattct cctgcctcaa cctcccaagt 3120agctgggact acaggcaccc gccaccacgc ccggctaatt ttttgtattt tttagtagag 3180acggggtttc accgtgttag ccaggatggt cttgatctcc tgacctcgtg atccgcctgc 3240ctcgcccttc caaagtgctg ggattacagg tgtgagccac cgcgcccggc ccgaaaatca 3300aactgtttta aagctgaagg cccccccatc ccatcctcta ccctccaatc ctaaggccct 3360ccttaacatt atgaaggttg agcaagtgtg gctcaaatct ctccctcctt ccccctcttt 3420ctccttctct tccttcctca ctccctccct tctttctctc tttccttctt tctctccttc 3480cttccttttc tctttccttc ccttttttcc tccttccttc attctttccc ttttttcttc 3540tttagaatag tgctgctgtg atcaccctta ggtgcctcca gttctaagct ttctgattac 3600ttcttccccc gaggtccccc agctcagcat ctgcccttaa aggaaaccta aataaacatg 3660tgttgttgca ttctgaatag cctctggttg atcaggagct tagactaagg attgaatagt 3720aaacggtgga tccaagtgat tgtgagaata ccacttggca gaatgtgatg tctggattgg 3780acatggagtg cataggaaag gacccgcacc tccctgggca tatgagtgtt tctggttgac 3840gctgtgcagc tgccgcataa caatgctggg aattatcctt tattcagggc ctgattgagt 3900gggggcgtgg ccaccccttt attgccttat tttttaatgt atatcattct ctatttacac 3960attgataatt ttttcctaca gggtaaaaaa aaaatgcacc agatgcagca cttttgtgta 4020agtatagaca catcagggct ctttgttaag gataagaaac aatcgcttcc acttcaggct 4080ctaatgttat catagccaac tgccagagcc gctctgtaat tacgtctcct gagtggaaag 4140ctctcaaaat gccacttggt ctaagagttt gaagctgggc ttgcctctcc cctttggcca 4200agatgactaa agcatcacat atttttaaag caacttttaa gaaatgaggt atagtgagtg 4260atgactagcc agcttctcgc tgctcacact gtagcatttt acacacattt tcccagggag 4320cccccaggaa cttggtgtcc agggatattt tccccatgtt ggggtgaagg ggctcacaca 4380atgtcacctg cacacgggcc tcacagttcc agatctgggc ttgtccactc tcccagctgc 4440ctgcctggga ggaaggcagg taaagttaag ggcagtgcca aggtttagga aatattgtct 4500gcctttggac caaatgtaat ttcaacttaa agtaacctag aaaatgtttt gcaaaaaatg 4560tgttttcaga aagcagaatt acactcagta tcccttcttt ttttggtttc tcaaccacat 4620ctttcattag agagatttag gacagctaag tagaaattaa attttccata tgtaaacaga 4680catggagaca gctggctttc actgggcaat agttcccggc tgggaatctc caatgaacaa 4740cttcgattct tgtattttta aacaggttgg ctttcctccc aaatgcaaat ataatgttct 4800acgtagaaat ttatatgtta ctgagaagca tcaagaagca aatgaaaatt cccggaagct 4860caccacccag agacggtcac tctcagggtt tcacacagcc tctctgccag gagctttcat 4920ttccttgggt cttgtctatt gttaggtcac atgttgagta ggatttgtct tggagcctga 4980acgctgacaa aggtgcacca cccttggacc ccatccaaag ccacacagga atgactactc 5040tggtcccacc cacttcctgc cctagctagt gtggtatttt tgtggctttc ccctgtcttg 5100tcagaagaat gagagtaact caacatgggc actttgtcca atccttgggt ctcaagggtt 5160ctgggaagga tccaagagct gtggttctcg gttcccaaaa gcaggcaggt gattggagaa 5220gcagccgggc tctgagtcct gtgcccgtgt ggcccaagtg actggctgtg accctaataa 5280gtgagcagaa gggcaggggc cggggtcacc ttgcacagga agtggcagcc atgagggtct 5340ctgcaggcca gagctgctcc ttcctcaggg gccatctccc aaggattctt gtgtcctagg 5400agccgctcag caaatggttg ttgactgact gagtgacggc actttggcct ctctgaacct 5460cagtgtcctg gtcattgaat gggactctta cttccaagct ctttactccc ctgagccctg 5520gcnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5580nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5640nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5700nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5760nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5820nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5880nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5940nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnngagc 6000agagggaaca gacgtgagag gcaaggcccg tgagggtagg gagaggtgct cccaatcatt 6060gtagaggcca ggaatgaggc tgggaggctg caagggccag gttccagccg ggccaactct 6120gagatgacgc tgaggattcc agctttactg tgagtcactt gtaggctcac tgcttctctt 6180aagaggcaag ggatggccag gcacggtggc tcacacctgt aatcccagca ctttgggagg 6240ctgaggtggg cggatcacct gaggtcagga gttcgagacc agcctgacca acatggagaa 6300accccatctc tactaaaaaa atacaaaatt agccaggcat ggtggtgcat gcctgtaatc 6360ccagctaccc gggaggctga ggcagcctgg gcaaagagag caaaactccc tctcaaaaat 6420aaaaataaaa ataaatagag ccaagggatg ccactagcta gttgtttaat taaacttttt 6480attttgaaat gactgtatag acacataaga gataatgcag agatactgtg ggcctttacc 6540cgggatttcc cagtggtaac agcttgcaaa acgatatagg accatatcat agctcggacc 6600ttgacactgg tacagtcaag acacagagca gcaccagcac agggagcctc ctgtggccct 6660ttgatgtcca cctgctccct gcctgcccct gacagcagcc aatctgttct ctgtttctac 6720aattttgtca ttttgagaat gttatataaa tggagtcata tagcatgtaa tcttttgaaa 6780ttatctttct tccaccagca taatcccttg ggattcctct aagggaattc atgcagggga 6840atcatcacac atctaggctg ctgtgtgtgt caatagtgtt tccttgttat tgctgagtgc 6900tgttgtgtgg catggatgta ccacagttaa tgtgtaaatg acaacatagt tttcatttcc 6960atgtctgcat gttcattgcc agtatataca aattaaagtg atttttgtga cttgattttg 7020tatccctgac cttgctaaat tcacatacta gttctgggag ttttgtttgt ttgtttgttt 7080gtttgttttc gcttcttggc attttctttg tagacagtca tgtcatttgc aaacatggac 7140aattttattt cttcctttct gctcagcatg cattttcgtt tctgttcttg ttttattgca 7200ctggctagaa ccttctccac tgtgttgaat aagagtggtg acagcaggca tccttgcctt 7260gttctcactt tgggggacta ttatgggcct gactatgtcc ctgcaaaata catgttgaag 7320ccctaatccc cagtacctcc gaatgtaact gtgttggaga cagggccttt aaagaggtaa 7380caaagttaaa atgaggtgct gagagtgggt gctgatctgt catgactggt gtccaaaaag 7440aagagctgag gactcacaga gggacaacca cgtgaggaca caaggatatg atggctgcct 7500acaagccagg gagagaggcc tcaggagaaa ccaaccctgc caacaccttg atctcagagt 7560tccagcctcc tggaatgtga gaaagtaaat ttttgacttt tcagccacct agccagtggg 7620actttgttat aataaccagg ataaactaag acactgcctt ccagagtacc acagggcacc 7680tctgttctga cttcagaaac tagttctgcc tgtctttgaa cttcatctaa cagtcttttg 7740tgtctggctt tgagatccat ccatataatt gctttctttt tttttttttt tttttttgaa 7800gacagagcct tgctctgtct cccagtctgg agtgcagtgg tgcaatgata gctcactgca 7860accttgacct ccggggctca agcagtctcc tgcctcagcc tctgagtagt tgggactaca 7920ggcacatgcc accatgccca gctctacatc attgttatat cagtggctta ctccttttta 7980ttgctggatc ctgttccatc catattatgg ctgcaccaca ctttgcttat tacgcattct 8040ccagttgtca gacacttgaa attgtttcca gctttgagtt atgaatacag ctgttctgca 8100catttgtgta caggtctttg tgtggacaga tgttttcttt cctcttgggt agatacctag 8160aagtgcaatt gctgggtcat agagaaggtg tatatttaac tttgtgagaa aatgccaaac 8220agttttccaa agtggttgta caattttctg cttccaccag catgagagtt tcacactgag 8280agttacgttt gctccacatc cttgccagca ggtggcattg tcagtctttt tcactgtagc 8340ggttcagatg gggctgtaat cattcctcag tgtggttttt agtttccctg atggcaggca 8400gtgttgccca ccagctcata tacttattgt ctatttaggt aacttaagtc aagtatatgt 8460tccagttttt ttttctcatt ttaaaaatgg aatcgtttgt cttttttttt cattgatttg 8520taggaatctt tttgtattct ggacacaaat cttttatcag atatatgtat tgcaaatgtt 8580ttctcccaat ctgtgatttg tctttgaaat tttcttttct tttttttttt tttttttgag 8640acagagtctc gctctgttgc ccaggctgga gtgcagcagt agcatgatct ctgctcactg 8700cagcctccac ttcccaggtt caagtgattc tcctgcctca gcctccagag tagttgggag 8760tacaggcgtg caccactatg cccagctaat ttttgtattt ttgtagagat gaagtttcac 8820tatgtgggcc agcctggtct tgaactcccg acctcaggtg atctgcccac ctcagcctcc 8880caaagtagtg ggaatacagg ccactgcacc cggccgcctt tgaaattttt aaaaaatatt 8940taattttttt ttttttttga gacagggtct cattctgcca cccaggctgg agtgcagtgg 9000catgatctcg gctcactgca acctccacct cccgggttca agtgattctc ctgtctcagt 9060ctcccgagta gctgggatta caggtgcgca ccaccatgcc caggtaattt ttgtattttt 9120agtagagaca gggtttctct atattggcca ggctggtctc caactcctga cttcaggtga 9180cccaccctcc ttggccttcc aaagtgctga gattacaggc atgagccacc ttgcccggcc 9240ttaaagactt aattttttta tagcagtttt acattcacag taaaattgag agaaaagtac 9300agagatttcc catatattcc ctgaccccac atattcagag acactctctt tatcaacatc 9360ctccccagag gagacatttg ttacaattga tgaacctgca ctgacacatc attatcactt 9420ggagtctata tgtggctggc cttttcacgt tcctaatggt gccttttgat aagcagaagt 9480tttctatgtt gatgaaacat attttcaggt tgaagaagtt gtttataaag ttttttttgt 9540gtgtggctta gtgcctttta tactctaaga aatcattacc aaccacaaat tcatgaatat 9600tttcacctat gtgtttttag aagctttaca gttttagcat tcacaattaa ggtttggatt 9660catttctagc tattttctgt gtatagtgtg agtaagcagt caaggtttgt attttggtac 9720ggctatctaa atgttccatt actatttgtt gaaaggacgt atctttccct attgatatgt 9780actggcacct tcattgaaaa tcaattgaac aaatgaatga aaatctatta gttcattgat 9840ttccattgat ttatccttat gtcaatacca cattgtcttg cttttgaaat ttattttatt 9900ttattattta tttagttttg aaatggagtc tcgctctgtc gcccaggctg gagtgcagtg 9960gcatgatctt ggctcactgc aacctccacc tcccaggttc aagcaattct cctgccttag 10020cctctccagt agctgggact acaggtgcct gccgccatgc ctggctaatt ttttgtattt 10080ttagtggaga cggggtttca ccgtgctgcc caggctggtc gtgaactcct gacctcaggc 10140aatccgccca ccttggcctc ccaaagtgct gggattatat gcgtgaacca ctgcgcccag 10200tcaaaattta ttgtattata gtaattattt tattaatgaa aataattact ataataaaat 10260tattttagtt aataaaatat aattactata gtaagtcttg aaatagggta attggttgaa 10320ctagttctct gttgttggac cttcagcaca tctctgacat tgggcaaggc tcggtgggtg 10380gctcatgcct gtaaatccca gcactttgca gggctgaggt agaaggatcg cttgagcctg 10440ggagttcaag gtcagcctgg gaaacatagc cagagtccgt ctctgcaaat aataaaaact 10500tagccaggca tagtgccaca cacctgtggt cccagatact caggaggctg agatgggagg 10560atccctcgag cacaggaggt cgaggctaca gtgagtcatg atcggtccag tgcatttcag 10620cctgggcaac agagggaaac tcagtctcaa aaaaaaaaaa aaaaaaaaaa aatcagccct 10680aataagactg tataaaatgt ccttgctcat ttgtgtgggt gttttggaag gtcgatggct 10740gaagcgagtg tcccttatgt ctgcatgtcc catgtccttg tgtccccatg cccccatgtt 10800tccaagacca cacgtcccca cgtcccctgt acccccatgc cctcatttct ctatgtccct 10860gtggctccac attcctcatg tctgcgtgtt ctcattcctc atgtccccat aacctccaca 10920tctccatatt cttcatgtcc ccgtatccct cacgtccccg agtccctgtg ttcccatgtc 10980tccatatccc catgtctcca tgtcctcatg ccccatatct ccatattctt catgtttcca 11040tgtcctcttt ccccattttc tcatgtcatg tccccgtgtc ttcatgtctt catgtcccta 11100tgtcttcagg ttcccatgtc cacttatcct cacatcccca tgtctccatg acctccactt 11160cccatgctct catgtcccaa aatgcccata tcctcatgtc ctctacgtcc tgttgtcatg 11220tctgcatgtc tgcatgtctc catatccctc gtgtccccaa gtcccccatc cccatgtcct 11280cattccccca tgaggacccc tcctatcccc acgtcctcat gtcacccata aaccagccca 11340gagcagagtt tgccttatgc tgtgtgcttg gtaaatatga gctgccttcc tccttaaacc 11400ttccattcaa aggctgcaga ataaccatgg gtaagatggc ttccctgaaa aaatgcaccc 11460caggtgtcac tactagacgt cagatggaca ctttccccca cagcggataa actggtagca 11520accctgggaa ggaatggggt gtaacagcca gggccagctg ggtgccgtaa cagtcccagg 11580cttagtcata gtgcagtgct ggcaatggga gagttgtggg ggctctgttc cttgtagtca 11640gatggggagc actcacccac caggacagag gcccagggag agcactcatc caccagggaa 11700gagcccctag gccagctgag agaaattagg ccaggggctg ctgaacaggg tagagcccct 11760gggcttccct cctggtaagt gggtgttccc ccttggcctc tctcccagtg ggtgatcccc 11820ccgggcctct ctcctggtgg gtgggccctc cacccaggcc tctctcctgg tgattgggtg 11880ctccccctgg ggctctctcc tggtgagtgg gtgctcttcc aggcctctct cctagtgggg 11940tgctccccct gggcctctct cctggtgggc gggcactccc cctggctgac agttctctgc 12000ccataggctc tctccatcct ggcctctgtg gtcccacaca ggcccctctg ggtcctggac 12060ttcagagctc ctctccctgg ccatggccag ctttcctgga agacaggact gggggtccct 12120gggacaaagc aggagtcctg gggaaacccc ctttctcatc agaggcaccc acccctcatg 12180aagtgagcca cctgggggag gatgagcagc acccttgagg gacaggctaa gggacactgg 12240aggcacaggg ccttctctgc agagcctctg actccacaaa gttattcaag ggctttgaga 12300ttgaatagca ccaatggatt gtcactgtgg tccccaggag ccctccatgg tcaccaggag 12360tcctccgggg tagaggactc atttgagcca ttggaacatg agtgaaataa ttggtcagag 12420aaattggaat atgtgttgat acttcctggt ggttgcttct ccactgtggg ctgatcccac 12480cattccccaa cttcaggtca cccttgccct ttttcatgat gaaaatacta catcacttta 12540aaacaatagg aaacacttaa ctctttagtt ttgatgggtc agtattgggg tgtggcttgg 12600ctatggcttg gggacctctc atgaggctac agatggtggc tggggctgca gtgtcatctg 12660aaggcctgac tgaaactgag gatcattttc aaggtgactc attcacacat cacaagttgt 12720tgctggctgt tggcctgggg ccttagttcc tctccgcatg ggcctctctg cagggctgct 12780tgagcatcct cataacatgg cagccagctc ccccacagtg agtgattcag caaggaaagg 12840caaaagctgc agtgttatgt atggcctggc cttggaagtc accttcatca tttctgtaat 12900gtcctattgt acagtagtgt ccacaggtca gctctattca cagtgggaga ggcagataac 12960cagcatttga tgatcactgg gccattgggg ggcaggctgc caaaaacatg tttctttttg 13020aatgatgtca ctcccaggtg gaggcaccac ttcctcccac tcaccccaac cagggacgac 13080aggacacttg gcctcagttc tactttcaag ccacaaaccc ctccccatcc tctttttgtt 13140ccttacctac cacaacccct cacctttcag gtcaccacag ccagcctgct gctgtgctgc 13200cagaatttat cttgaccctc ttaggacgac agttgtgtat tcctagtgta cctgggtttg 13260tgtccctggt cctaatggga ccccactgct ggccttctgg cacaggggac ctctgtagag 13320gagaaggctg aaccaggtgg tggtggcagg cactgctgtc cttcctggcc tgggctggaa 13380gcatctggat ggtccagggc tgcctcctca gggggcagga gccaagactc cagggccccg 13440gcattgacag cacagcaacg gtgattcaga tgttggtggc tgggatccct tgagtaccta 13500ccataggcaa agccctcatt tctcacaaaa actccaccac atgcatccca cattttctcc 13560atttcatggg aggaagaatg gagtcttcaa gnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13620nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13680nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13740nnnnnnnnnn nnnnnnnnna aaaaattagc tgggcattgt ggtgggcacc tataatccca 13800gctacttgga aggatgaggc aggagaatct ttgaaaccag aaggtggagg ttgaagtgag 13860ctgactttgc gccactgcac tccagcctgg gcaacagagt gaggctccgt ctcaaaaaaa 13920aaaaaaaaaa gaaaagaaaa gattaagttc agcaatatgg ctgaggccac agcacccatg 13980cgtccgagct caggtgacac tgtccacccc atccttgtca cggctgggtg tagctctgca 14040tcctcacaca ggcagggaac ttagggacaa actccacatc acctcagcct ttagcttttg 14100ggagggaggg gcacaccact gtgtcctgag cctcagtttc cctgttgtca gacggggata 14160accatacttc acaggggcat gaaggggccc aatgagtaac cattcacagg gcacctgcgg 14220cagcacctgg cacagggaaa ctgaggccca gagaaattgc ttttatttat ttatttatta 14280ttttattttt attgagacag tctctttctg tcccccaggc tggagtgcag tggcgcaatc 14340tcagttcact gcaacctcca cctctggggt tcgagcggtc ctcccacctc agcctcctta 14400gtagctggga ttacaagcat gcgtcacaat gcccagctga tttttgtatt tttagtagag 14460acagggtttc accaagttgg ccagactggt ctcaaactcc tgacctcaag tgatcctccc 14520tccttggcct cccaaagtgc tgggatttca ggcatgagcc accatgccca ggtgagaaat 14580tgctttaaaa aaaatgaata taagcctgga ttaacaagca ttattaaaat tgagtcagac 14640tttgcactct aagcccttca ggacctggtg ttattattta ttgattctct ccctaaaaca 14700atgagggtgc cctcctggtg gggctgctca gcggggcagg tgctgggcct gctggagcag 14760ccctcgtggg tcaggtctcc caggaagcat cttgaaatat ctgtttgcag ggggcctctt 14820caccttctaa cttgtggcca tttcattgtt cccaaccaag agggtctgag gaagcctctg 14880atggtgtgtt tctgggctca ctctgggctt gtctggtcta gggccggccc cctcattccc 14940tgctgggccc ctcctccctg aagatctaca cagcagatct actcacaaaa gtccacccag 15000cagaagcagg aggatggtgg ttgtgggtcc tctgtgagca gccctgttaa cctttaaacc 15060agcacggccg ccgcagctgt gagcctagca cctgatcagt ggagagctgt ggattgcatg 15120tttgtttgcc attgcccccg ccaccctgca agttgcacct tctagaatca gcaagccaag 15180ctcctctcac ccagcgtaat gatgcggaaa tgcaaatgca ccatcatgtt gtgacccata 15240ttgcgaaaat tagaaaaaag gaagttgtgt ttcgctattg cacgaagttc agcccagagg 15300agaaactcgc tcgccttcag aagacaggtg agtgggcttg gtcctgaggt gttggccgag 15360ccctgagatc ctaactgctt ttgtggaact ttggggtggt aggaacattc agggcaggag 15420ccatttgagg gagacggatg ttttcaaggc aggaagtggc tgtggtggca cggaatggtg 15480tcttgttttt gttctgttgt ctttctctgg gttcctgtag catgatgtgt aaaaattcag 15540agttattgtg ggcctcgaat ggcaagtgtg aaaagccttt gtaaactgct gagtgctggg 15600acagtgttgc ctatcgtctg ggggtggctt aggctctcac aagatgaacg gtgcctgccc 15660gtgtccccat cgaaggctgg caggggcctg ggtccgaggc tgaccagatg cagggagcct 15720ttttccatcg gctggcagcg caccagccag ggcatcgaga ccacggccag cccctctcat 15780gtcacccagg cctggggggc ggggggtagc gcgagccttc ctctgcctga acagaggagg 15840gtggtcccag acgtgtcctg ggggctaggc tttcccacac agtgtgtctc cagaagcttc 15900ctctgcactc tgcaggttag atattagcag atgcgggcct gtgtagcatg gggctgtgag 15960gaaggagtgc tgcgcccacc aaagcctcct cagcaccccg tgctgtgcct ggcagtgtgg 16020gacagtggcg gggccacctc aagaggtgca tggactgatg gcagggtccc agcggcagtg 16080gcggcacaac ccgatgccac cgtgattgtg cccactgtgc agagggggcc cgaggggcag 16140aggcatgttc gggtcacaca gcatggccac actcatctgt aaagtggtga ctgagtcgtg 16200ccaggctcac tgtagttcgg ggctggacgt gcttagcagg tgccactgcc attcagatcg 16260gtgcttgcca acacccaggg cggatgctgg tcagccattc ttgcagggga ggaaaccaag 16320gcacacagct gacttgccct gggcccccca gcctgcaaag tgtctgactg agccccagta 16380caaatcttca caacttgctc agtggcccca gagccgggtg gggctgacca gctgggtgtg 16440aaagggagga agtggtttgt ccccagctgg gctgttacag gaggccagag gaggccccag 16500ttccactcct cagggggcgg aggtccacgt ctacacaaaa accagcacca tgcagtgtgc 16560aagacccctt ggaatgacac gaggccgagt gccctgcaca cagaggcctg cagccgtgtt 16620gtacagaggt tagagctgaa atgacctttt cctgagaagc ctcaccactg tccgaggatg 16680ggcctgacct tcctggcctg tgccccttga ggactcccag gttagtccct gggctagaag 16740agtgacctgg gagtcactgg tgcatgtgct gggagctccc aaagcatcct ccccacctgc 16800cccgataggt ctatcaggag cctggtttac agataaggaa actgaggccc ctcagtaagg 16860gcagtgctgg gcaggtgtga aacctagaca aggatgagac tgggcttgtg gaacaaggag 16920gctcggaggc aagcatgtgc cgaaaacaat gaacaaggac aggaggaaga gcagagccgg 16980gcactgtggt gcctgcctgt ccctaggcgg gacgaccgtc agctgtcggg gctgagccgg 17040tcgggaagga aacaggatat gaggaccttc attcatccct tacatttctc cccgtgtcct 17100gccttcttct ctgcagatat tgtgggtcct tgtaggtgac aaggggccca ttgtctgagg 17160agggagacag ggacatgagc ctgccacggt ttcatctggc tagctgcaag gacagtgcag 17220ggcaggccct gtgtgaaatg ggggcagcca catctgggtc tggggacaag ctgcctgctg 17280cctagtgaca cgaacctgtg cccgtggtgc ctccgtcccc acacgtgagc ttccccactc 17340cctccttctg agcttgctgc ctgccggtga gtgtccctgg tctcaggaaa gtaacccaca 17400ggcaaatggt tgctgcattt tcccaaaagc aagtattgaa cccctaacaa taatctcagc 17460cctccaggac cccctgctct gtgccacaag gtgtgctgtc ccccagcccc accctccagg 17520tgggggcacc gcagactcag gcctggaggc tgcacctgtg ggccctgccc ggggcccacc 17580ccacctggga gtctgtatga ggcctctgcc cagggtgcca gtcttctgag gagcagcggt 17640ggctgtgcat gtggaccaag gaaggagggc aagagtgtgt gaatgagggc tgagctccgc 17700agcctcagaa agggacacta aggatcccgc ctcccctgga cacccccagc tgggcaggga 17760ggttgacaag gaggctggcc tggtacaggg ccttgccctt cgcaccttgt cattttggtg 17820caaattcaaa aaacttctca tgagaatctt ctgcatatgt ttaaaaaaag ttttcttttt 17880aaaaatggca gtaaaataca cataacctaa aacatagcac tttcatcttt tttctttttt 17940ttttttttgg ggggggatgg agtctcactc tgtctcccag gctggagtgc aatggagcca 18000tctcggttca ctgcaacctc catctcccag gttcaagcga ttctcctgct tcagcctccc 18060aagtagctgg gattacaggc acccaccacc aagcccagct aatttcttgt atttttagta 18120gagagggggt tttaccatat tggtcaggct ggtcttgaac tcctgacctc aggtgatcca 18180tctgccttgg cctcccaaag tgctggaatt acaggcgtga gccaccgtgc ccagcctcct 18240tcatcgtttt taagtgatca gtacagtgcc ctcagcacat tcatattgct gtgcaaccat 18300cactgnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 18360nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnaaa actctgtccc 18420tgttaaaccc tctccattcc ctcccctggc cctggcagcc accattctgg tttctgtctc 18480tgtgaatttg actcctctag gacctaacgt aggtggaatc acacaggatt tgtccctttg 18540tgactagctt atttcactta gcataatgtc ctcagggtcc acccctgttg tggcatgggt 18600cagaatttcc ttccggttta tttatttact tatgagatgg agtctcactc tgtcaccagg 18660ctggagtgca gtggcgcgat ctcagctcac tgctacctct gccttccagg ttcaagctat 18720tctcctgcct cagcctccca agtagctgag actacaggca tgcaccacca tgcccagcta 18780atttttgtat ttttagtaga gacagggttt catcatgttg accaggctgg tctcaatctc 18840ttgacctcgt gatccgcccg cctcggcctc ccaagatgct gggattacag gcatgagcca 18900ctgcgcccgg cctccttctg tttaaggctg aacatgattc tgttctatgg agaggccacc 18960ttttctttat ccaggtttct agggatggac acttgagggg cttccaccct ttggctgttg 19020tagggaatgc tgctatgtat gtgggtgtac aagtacctgt ttaagaccct gctttcaaac 19080cttttgggag aaacattctt gactagatgg actcacatgg gaaactggtg ttggtttaaa 19140tgtggctgcc ctaagaaaag ctgccgcatc cttgggagag gtagataccc cccgcccacc 19200agcccccgat tccaggctgt ccagcctctt ctcccagtcc acagtcacag cgtcatgcca 19260gggctatggg tcatgaagac tcatacagtc ttaaacagta gaagtaactt gttaaggtgg 19320cggtgtgagc catatttgtg agtgtctgct catggcatga ctacaaacgg ggaggtgctg 19380cgttgatctg cctcctcttc tccctgctca gcttgccctc acttcctgcc ctctccgagg 19440cccctctgac ccccatccca attcaggagc atgtagcacc tcactgagtt ctcagcagag 19500atgatggaga cccctcctcc atccactccc acagcagaca gggcatttcc caggctgaga 19560ccccaaggta attactctgg gcttccaggc accgctaggt nnnnnnnnnn nnnnnnnnnn 19620nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 19680nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 19740nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 19800nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 19860nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 19920nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 19980nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 20040nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 20100nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 20160nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 20220nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 20280nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 20340nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 20400nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 20460nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 20520nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 20580nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 20640nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 20700nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 20760nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 20820nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 20880nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 20940nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 21000nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 21060nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 21120nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 21180nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 21240nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 21300nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 21360nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 21420nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 21480nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 21540nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 21600nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 21660nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 21720nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 21780nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 21840nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 21900nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 21960nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 22020nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 22080nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 22140nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 22200nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 22260nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 22320nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 22380nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 22440nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 22500nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 22560nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 22620nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 22680nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 22740nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 22800nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 22860nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 22920nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 22980nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 23040nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 23100nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 23160nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 23220nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 23280nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 23340nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 23400nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 23460nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 23520nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 23580nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 23640nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 23700nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnntg tagatttata gtttccatca 23760aatctagaaa acttgtcatt atttcctcaa attttttttt gtccacccct ttttgagact 23820ctgatcacac atgtattaca ctacttgaag ttttcccaca tttcactgtt gctcagtttc 23880tctgtgtgtg tgtgtgtgtt tcattttggg tagtttctgt tcctgtctct tcaaattcac 23940tagtttttct tctgcaatgt ctaattggtt gttaattcct tccagtgtgt ttttggtctc 24000acacttcgta gttttcatct ctggaagttt aatgtgggcc ttcttacatt ctccacgtct 24060ctactggatt ttagaacata ggatgacaca gacatgatag tagtttttaa tgtccctttt 24120tgctaattcc aacatctgtg ttagttatgg gtcagtttat attgaatttt ttctcttcat 24180tagttatagt cccttttatg cctggtaatt tttttttttt tttttttttt tgagacagag 24240ttttgctctg tcgcccaggc tggagtgcag ttgtgcaatt tcagctcacc acaacatccg 24300cctcctgagt tcaagcagtt ctcctgcctc agcctcctga gcagctggga ttacaggcac 24360gcaccaccat gcctggctaa tttttgtatt tttagtagag atagggtttc tccatgttgg 24420ccaggctggt ctcgaactcc tgccctcaag tgatccatct gccttggcct cctaaagtgc 24480tgggattaca ggcgtgagcc actgtgcccg gcctctgcct ggtgattttt gattggatgc 24540cagactatgg gaattttacc ttgccatgca ctagatattt ttgtagttct ataaatattt 24600tttagctttg ttcaaggatg cagttgggta aacatatgga cttcttcctg ttaagttttg 24660ttaatatttg ctaggtggga ccagagcagt gatcactgca gggctaatta tccatcattc 24720ctgaagcaag accctctgag tattcaacgc aatgccccgt gacctgtggg gcttttaggg 24780ctggctggtg ggcataggcg tgtccctgcc ctgtgaaagc tccctgtggt cctcacaggt 24840ggttctttct ctggcctcat gtgctctccc caccgcacac atgctgaacg gtcctcgagg 24900agcccctctg agatctctgg gttctctctc tctctgcagc tctctcatct tcagggctcc 24960atgctgtgga ctctggccac cttagtcttc tgggcctccc agttttgtct tctcaactta 25020gagagtccac tgggccccac agggcttccc ttccctgatc catggcccag aaatggtctc 25080cgggcagtga gctggggcaa taggagggct cacctcattc attcccgtct ctcagggacc 25140acagtgtttt accgcttggt gtagagcctt gcaaacttgt ttcgtaggtt ttgtccattt 25200ttgttgttgt tccaggtgag agtaaatctg gtttctatta ctccatcctg accagaagca 25260taattccccc gagatggggt tgaaccaggc tctgtcttgc cagacagcta cctttgaaca 25320caccgaggct gagcacagct cacattccat ctggtgtatt tttattgctc tgccatgttt 25380gacctctgga aacctgagtt tatctcttgt gctatgagaa gaatgcagca aaacatgtat 25440tttctgttta gaaatcttaa tattgccccc cattgaactc gttaggaatg cggcagtagc 25500caatcataaa tgcccacctg agcgattctg gagtcattta aagaaacgtc cactcaaaag 25560gggttttgcc ccaagacagt gagcactcag gagactgtgt gtggtggggg tgctccacca 25620tagagctggt cggcatccac agccaccagg acgctgccag agcccaggcc tgcgccacgc 25680cctgcccaga ggcacacctg cctgctcaag gcaaccaggg caggctcaag gcagccaggg 25740ccagctcact gtgtgcctgt cactgtgtga agcctgcttc ttctttattt cgcttagcaa 25800gtgaggcagg agcatgtaaa gctgctggca gaggaatatc taattgtgag tgtgagtgcg 25860tgtgtgactg tgagtgtgag ccagcccgtc agtgtgaatg tttgagagag cctctctgta 25920tgtgcctggg ggagggtgtg aggtgtgggc tagggagggg cctggcgatg cccaccggct 25980cacctcacac tggacagtgc ctgcatgttc aagatgaccc ttcccctggg ccgcaggatg 26040ctgcatggca agggacaagc tcagccctgg ccccagagtt tccagccgag caagacactg 26100cccaccctac agtgcatggc tgctggtgca ggccccaccc ttttggaggc attgtgtgcc 26160agcctggggg ctgaggatat gactgtgcag atggccatgc cccaatcctg gagtccacca 26220tgctgggtgg tggcatggag cttccctcct ccgcaccccc gcagagccgg ggcctctggc 26280tgctccaaat gccagttgga gaaggtggtt ttctttctct actaatgtct tttcagtacc 26340tcctaaatgg ctctactttg aacctgctgg gcaaggaaga gattttcaag gaaaccatct 26400accgtgtgca agctcctgcc ggccaacccc agaccccagc acggagccag gcgcctgtgc 26460ccgccaaccc tcagcatcct cctcagaaag gctggtggca tcaggtatgg ggataagtga 26520ccgccacttg cagcccctag gctcctcggc agctgcgtct tgggttcaag aggatgattt 26580tgagggatga cagtctccct ctaggcccgt ctcacctttg ctcctcctca gaggcgcccg 26640gtgaattttc tgtgcactta taattgacac ttgaatttgg tccatgagag gcgtggtggg 26700ccagtgtgag gtggctccag tgcacaggag ggaggaggag ttttggggca aagaccttgg 26760tccacatcct ctggcaagac gcacctcctg ccccagccct cccctgaggg agatagtaga 26820gacagctggg accctggacc cctgggggct gttttctgac ctgtccagca ggcagtggtg 26880aggaggagga ggaggaggac accggcctca tgtcctggga gtatcagagg attgaaaaag 26940caccacggat gcgctataaa cagaagccac tgtgttcctt ttggttatgg tcactacctt 27000gagcatctat tttgaattta ttgaacagca gatagcttca caccacaggg ttaatttttt 27060ttatgcggaa caaacaattt taaaacaaaa cattttccat tttctgttct ttaaaagcct 27120gtctgtacaa gacccagctg aagctgacca gggcattcac acttcacaca caacccccag 27180cccacctgct ccaggctttg ggtggggggc ggagggtact gcctgggagc tgcatttggg 27240ggttgcacct gagggtgctg gtccatgcct ggctagcact gactctggct tgtttctccc 27300ctacaggaag cccctggcca gcctccacct gagcccagtg agctcagctt taaggatgga 27360gtcaggcagg gggtcctcaa cccctccagg acccattgct gccctaggga tgccagacac 27420tgggcctggc agttcctccc tagggaagct tcaggcgctc cctgttgggc ccagagccca 27480ctgtggggac cctgtcagcc tggctgcagc aggggacggc tctccagaca taggccccac 27540gggagagctg agtggtagct taaagatccc caaccgggac agcgggatcg acagtccatc 27600ctccagtgtg gctggagaga actttccctg cgaggagggc ttggaggctg gcccaagccc 27660cactgtactg ggggcgcacg cagagatggc cctggacagc caggtcccga aggtcacccc 27720ccaggaggag gcggacagcg acgtgggtga ggaacctgac tctgagaaca ccccccagaa 27780ggctgacaag gatgccggcc tggccccagg taggcttccc cttctctgtc cccgctgcgg 27840gctgcaggcc tttccttagg aggggtttga gattttcagt gggcgtcatc cccacacctt 27900gtttccagga atgtggcaca caggctcgtg ctgtgtgcaa ggggaggtcc tgggatgcag 27960tctgccctag aaaagcctga gggaaaaatc catgcaggcc aggccaagcc cacctgacac 28020cttgagcagt gacctcagtc ctgagtctca gtttccacaa ctgcacagtg ggcacctccc 28080gaccctggga aggtatgatg gtcagtccta ctggtggtgc tgccaaagct gccctttgca 28140cagtgctcac tgctgacttt atgaaactcc tgggaggaga cgatgtgagt ggggaaactg 28200ttttcctcaa tgggaaactg aggcacagag ggcatggtac ctggccagcg tcacctatgg 28260ggtgtggcac aggctgggtg ccctgtgctc ttcaggacat ggtggggcag gacctgtgag 28320gcccgcagtg cagtgtggag accagagcag acagttccca caacacgatg gggctgcatg 28380gagggagttt gtttgcgttc tcattagcgt ttatggggct cttggctcgg tgctgggatc 28440ttggggagat ggagaggcag cagatggcct caggctcaga gaaactgtgg gtatgggggg 28500cacagcttgg gtaagttgcc aagtgaccag gtcctgggag cgctttgcgg gtcaggcttg 28560ctggctccta ggtggggacg ttgctggctg acacggggac catcgggaga cgggctgggc 28620aggtgaaggg cagtccggtg cctggaccac tgtgagctgg cttggcggag acgaggccag 28680ggctgtgttc aatggaggct ttattatcac tcaggtgtag gaaggatcag accactgcca 28740ttgacaagat ggtttgtcat cctcagtttc caagagaaag gggcatgtca caccacaccg 28800tgaggccaca gggaagccca gggtcagtca gaaggcagag ggttcgaggg ggaactgtgg 28860gcctgagacg gtaatgtggt ttctgcggga aacttgagtt cccagccatg atgggaatgg 28920gaggtcagac acacctgatt ctactccctc acttttgtag tttacacaca actgaccagc 28980attaatctta aaatagagat cagaagactg acagaataga ctctgtggca ataagatacc 29040aaattataaa caggaccagg caagtgtaca caccatacac ttaaagaata aactatattc 29100tggctgggcg cggtggctca cgcctataat cctagcactt tgggaggccg aggtgggtgg 29160atcacctgag gtcgggagtt tgagaccagc ctgaccaaca cggagaaaac ctgtctctac 29220taaaaataca aaattagctg ggtgtggtgg cacatgcctg taatcccagc taattgggag 29280gctgaggcag gagaatcact tgaacccagg aggcggaggt tgcggtgagc cgatatcaca 29340ccattgcact ccagnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 29400nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 29460nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 29520nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 29580nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 29640nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 29700nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 29760nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 29820nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 29880nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 29940nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 30000nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 30060nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 30120nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 30180nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 30240nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 30300nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 30360nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 30420nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 30480nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 30540nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 30600nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 30660nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 30720nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 30780nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 30840nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 30900nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 30960nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 31020nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 31080nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 31140nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 31200nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 31260nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 31320nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 31380nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 31440nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 31500nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 31560nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 31620nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 31680nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 31740nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 31800nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 31860nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 31920nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 31980nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 32040nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 32100nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 32160nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 32220nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 32280nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 32340nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 32400nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 32460nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 32520nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 32580nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 32640nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 32700nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 32760nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 32820nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 32880nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 32940nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 33000nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 33060nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 33120nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 33180nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 33240nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 33300nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 33360nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 33420nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 33480nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 33540nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 33600nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 33660nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 33720nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 33780nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 33840nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 33900nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 33960nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 34020nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 34080nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 34140nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 34200nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 34260nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 34320nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 34380nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 34440nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 34500nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 34560nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 34620nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 34680nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 34740nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 34800nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 34860nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 34920nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 34980nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 35040nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 35100nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 35160nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 35220nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 35280nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnataaag 35340gtgaattcag aggtcaggta aatattaagc aaacacccat ctaggaaagt atatttttgc 35400cccaaagagg tgtagggtat ttagacacta ccagggactg gtgggaaagt ggtaattggt 35460ctcttaaata atgtaaaggg gtgtgaatct tttttttctt tttttttttt ttggagggag 35520ggagtgctat ttgcccacat tacctgactg catttggaga agaattgctc agaggagatt 35580agcacagagt aggcagctct tgaacccaaa gggaaattta taattttatt tgctgcctcc 35640agagtcaccc ttggctttgt cctgctagtg ccaatgtctg atttggaagc cagctagagc 35700agagagctct ccagctcaag gccatcaggg gttgggattc tgtcctgggg gcccttcagc 35760cttcaaagca gtcccgtttc cagtggccaa gctcttggca gagaggggaa accatgtgag 35820gtttcctccc gtttgtccca ctggggcagt ttgccttcca gtggcctggc ttcccacgcc 35880aatggcagtt acctggagga gtgtccttag ggaaacctgg aggaggctgg tgggcttgca 35940gagcagccag tagttaagcc tgtggcctgt agttttcttt tttgttgtgt cttttcctgg 36000ttttggtatc aaggttatga tgaccttgta aaatgagtta ggaagaattt cctccgattc 36060aattttttgg aatagtttga taattggtat caattgttct ttataggttc agtagaattc 36120agtggtgaac ccactgggtc ctggactttt tttgtttgga gatttgtttg tttgtttgtt 36180tgtttgtttg tttttgagag agtctcaccc tgtcacccag gctgggagtg cagtggcgca 36240atcttggctc cctgcaacct ctgcctccct agctcaagct attctcttgc ctcagcctcc 36300tgaggagctg ggattacagt cacccaccac cacacccagc taatttttat atttttttgt 36360agagatgggt ttcaccatgt ttcccaggct ggtttcaaac tcctaggctc aagcagtatg 36420ccagccttgg cctcacaaag tgctgggatt acaggtgtga gccattgcac ccggccctta 36480aatttcattc ttaatttctt ccttcatcca ttcatcattc aggagcatgt tatttaattt 36540ccatatattg atatagcttt gaatgtttct cttgttgttg atgtctagtt ttattccatt 36600gtggtaagat aagataccta atataatttt gatttaaaaa aaattctgag gcttgttttg 36660tgtcctaaca tattgtcagt cctggagaat attccatggg tacatgagaa gaatgtgtat 36720tctgcagccg ttgggtgaaa tgttctgtaa atgtctgtta ggtccatttg ttctatggtg 36780tagtttaaat ctgatgtgtc tttgattttc tgtctagatg atctatctaa tactgagagt 36840ggggtgttga agtctccaac cactattgta ttggaggtct atctttccct ttatatttaa 36900taatatttgc tttctatatc taggtgctct agtatttggt gcatatatat ttgcaattat 36960attctcctcc tgaattaatc cctttattat tatgtaatgt ctttctttgt ttctttttat 37020agtttttgac ttgaagtctt cttcgtctga tataagtata gctactcctc cttgttttgg 37080gtttctgttt acatggacta tcttttttca tccttaactt tcagtttatg tgtgtcttta 37140caggtaaggg gagtttcttg tggacagcat atagttgggt cttggtttgt taatccattc 37200agctagttta tcttttaaac aggggaattt aatctattta cattcaagat tattattgat 37260tggtgagaac ttactcctat cattttattg attgtgttct ggttgttttg tatatccttt 37320gttacttctt ctcattattt atttttgtag ttgggtggtt ttctgcagtg ataaggtttg 37380attcatttct tttatcattt gtgtatcagc tttaccagtg agttttatag ttttacatgt 37440tttcaagatg gtgtttattg tcttttcact tccagatata agactgtctt gagcatttct 37500tataagactg gtctagtggt gatgaattcc cttagatttt gcttgttggt taaagatttt 37560attcctcatt cctttctgaa gaatagctgg gaggattgct tgagctcctg ggttggagtg 37620gcagactgtt cttttagctg aaagtgtggc agtgagggtt ggtttccctg ctgtgcagga 37680acagaatcac agctgatcct gggcccaagc tctatgcatc tggggttgtg gcattcaagc 37740actcatgtgg tcttggtgga atgcagacgg agcccagtgc tagagaggta cagtagctac 37800tgcctggagc attctggagg ttgctctggt ctcaggatgg tgccatgcca cagcagcctc 37860gttcacagtg ggtggttggg gggtggggtg tgtacacttt gtgatcctaa tccgggtgat 37920gtagctatgc aaattctcac aactctccaa actgggcttg gggcttgcga ggactgtggg 37980attctcttgt gttatacttt ttccctgaaa agggaagtcc ctcctgactc tggatagatg 38040ctatctggtt ggagggagat gggaccacta gaggctgggt gcctccacac tgctctctca 38100atcatcacag gtgcatctct actccaccac tcactccacc gccagccaaa tcttagttgt 38160ttgtgcctta gtcttttctt gggatgaggg gaggaatgcc aggtgtttct agtcagccat 38220cttactgata tcactctcct tctttttttt ggtacaaggt ctcactatgt tgcccaggat 38280agcctcaaac tcctgggctt aagtgaccta cctcaacctc ctgagtaggg gggactgtag 38340gtgtacacca ttgcatccag ccttgtaaca gtttttgcta taaagtctat tttatctaac 38400tatggccact cctgctgtct tctggttacc atttgaatga agtatctttt tccatcctgt 38460cactttcagc ctatgtgtgt ccttatatct aaagtgagtc tcttctcaac agcatatagt 38520tggatgctgt tatttttaat ccattcagcc aatatgtctt tttctttttt attattatta 38580tacttcaagt tctaaggtac atgtgcacaa tgtgcaggtt tgttacatat gtatacatgt 38640gccatgttgg tgtgctgcac ccgttaactc atcatttaca ttaggtaaat ctcctaatgc 38700tatccctcct ccctcccctt accccatgac aggccctagt ttgtgatatt cctcactctg 38760tgtccaagtg ttctcattgt tcaattccca tctatgagtg agaacatgcg gtgtttggtt 38820ttccgtcctt gcgatagttt gctcagaatg atggtttcca gcttcatcca tgtccctaca 38880aaggacatga actcatcatt ttttatggct gcatagtatt ccatggtgta tatgtgccac 38940attttcttaa tccagtctat tattgatgga catttgggtt ggttccaagt ctttgctatt 39000gtgaatagtg ctgcaataaa catacatgtg catgtgtctt tatagcagcg tgatttataa 39060tcctttgggt atatgcccag taatgggatg gctgggtcaa ataatatttc tagttctaga 39120tccttgagga atcgccacac tgtcttccac aatggttgaa ctagtttaca gtcccaccaa 39180cagtgtaaaa gcgttcctat ttctccacat cctctccagc acctgttctt tcctgacttt 39240ttaatgattg ccattctaac tggtgttaga tggtatctca cggtggtttt gnnnnnnnnn 39300nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 39360nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 39420nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 39480nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 39540nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 39600nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 39660nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 39720nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 39780nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 39840nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 39900nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 39960nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 40020nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 40080nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 40140nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 40200nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 40260nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 40320nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 40380nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 40440nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 40500nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 40560nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 40620nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 40680nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 40740nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 40800nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 40860nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 40920nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 40980nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 41040nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 41100nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 41160nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 41220nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 41280nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 41340nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 41400nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 41460nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 41520nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 41580nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 41640nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 41700nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 41760nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 41820nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 41880nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 41940nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 42000nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 42060nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 42120nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 42180nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 42240nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 42300nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 42360nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 42420nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 42480nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 42540nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 42600nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 42660nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 42720nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 42780nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 42840nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 42900nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 42960nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 43020nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 43080nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 43140nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 43200nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 43260nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 43320nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 43380nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 43440nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 43500nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 43560nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 43620nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 43680nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 43740nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 43800nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 43860nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 43920nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 43980nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 44040nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 44100nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 44160nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 44220nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 44280nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 44340nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 44400nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 44460nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 44520nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 44580nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 44640nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 44700nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 44760nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 44820nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 44880nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 44940nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 45000nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 45060nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 45120nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 45180nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 45240nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 45300nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 45360nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 45420nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 45480nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 45540nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 45600nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 45660nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 45720nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 45780nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 45840nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 45900nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 45960nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 46020nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 46080nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 46140nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 46200nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 46260nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 46320nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 46380nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn ntctctttta tctcattgag 46440catctttatg acaactattt cgatgttttt tctcaagcag ctcatagatc tgtgtttcct 46500taagatcagt tttgaagttt tttattttgt tcctttgatt gggctacatt tttctgtttc 46560tttgtagcta attttgtttc atctagaagg tcagaagcaa aaaatgcttg ctaagtctga 46620ttaaaagagc cccagtacct tcaaggcctg tctactgtgg tactggagtg attatttcta 46680tcttatcttc tttacagctt ggtctggaga gctgccttag actctccaat gaatctattc 46740aaatagctgc ctctgttacc ttgacttgtc tcagattccg ttgacccaag atgggtcctg 46800gcactaggaa tataagactg cttccattat tttgacttgc tccaggttag ggagaagatt 46860atgcaaggct cctactgact gtgttttatt tctagctttg atgtctgggc actgatttcc 46920ctaggtttaa ctacttgctc catgttaagg ccatgctgtg gaaatctgtc tgtgtgactg 46980tcatgcaggc ctgtctatgt gattgtcagg gagaattggc ctgccacaat agggccttca 47040ggaggtgatt aagtaatgaa gtcagagcca tcatgaatag cattagggcc tttataaaag 47100agaaagttca tgggtttgct ctctgctctt ccaccatgtg aagacacagc cttcatctcc 47160ctttttctct tctactgctt ccactatgtg aggggatagt gttcaggcca ccatcttgga 47220agcagagatc aaccctcaga agtgtgagaa ataaacttct tttgtttaga aattacccag 47280tctcaggcat ttcagtgaaa acttccatag tgatgatttc cagctttaat tagctcatac 47340tctgtgccct gtcctgcatg aagtgtttca tatgtgttgt tccactccat cctcataaca 47400tccctgtgat gtaggtacta ggtggtgacg ctgctgcttt gcatttgggg tcactgaaac 47460ttacagaggt ttgttagcct gcccaatgtc aaaaacagga ggaggccagg cacggtggct 47520cacacctgta atcccagcat tttgggaggc tgaggcgggc agataggttg agtccaggag 47580ttcaagacca gcctgggcaa catggcaaaa cctcatctct acaaaacgaa aatagaaaaa 47640aactagctgg acatggtggt acgcatgtag agcaggtaaa atggactaag acaagcttgc 47700caaacatcac caatttccct acacctttta ctgaaatctt ttcttggctt tataatggct 47760tgggtgctgt agcttctcgg ctggtctctg gaattctcac acaggtgctc tggcccatat 47820attgttgtta accttatgtc tctgagcaga aggagggccg agagctgttt agtccatcat 47880cttgctgaca tcactgtgca atatattttg acgtggtcgc tgcaactgct ctgtagtcaa 47940aggcccatct atgagaactg agcaccacat cgccatcaca gtggcccctc ctgccccttc 48000tcttgcccca gtggcctcct gccacccctg tggcccctgg ttctcctgca cacagtggca 48060gcccgttagg tgccgattcc ccaaggctca tcttccccag gtgagctgca tctcttctct 48120ccgcagcact ctggccccca gaagcttctc cacattgccc aggagctcct gcacaccgag 48180gagacctatg tgaagcggct gcacctgctg gaccaggtag cccacatggc ttgggggcag 48240tttcagtatc tcttagcatt ggctgggcat tataggtgca gtgtgaatga cttaaatact 48300aaaactcaga caaatttaag tctggtctac aaactttttt ttttttttga gacaaagtct 48360cactctgtca cccaagctgg agtgcagtgg cgcgatcttg gctcactgca acctccgcct 48420ctccagttca agcaattctc ctgtctcagc ctcccgagta gctgggacta caggcacatg 48480ccaccacgcc cagctaattt ttgtatttat agtagagacg gggtttcacc atatttgtca 48540ggctggtctt gaactcctga cctcaggtga tccaccagct ctctacactg gttattctag 48600ttagctattc gtctaacctt ttttcaaggt ttttagcttc tttgcgatgg gttcaaacat 48660cctcctttag cttggagaag tttgttatta ccaatcatct gaagcttact ttcgtcaact 48720caccaaagtc attctccttc cagctttgtt ccgttgctgg caaggagctg tgttcctttg 48780gaggagaaga ggtgctctga tttttagaat tttcagcttt tctgctctgg tttctcccca 48840tctttgtggt tttatctacc tttggtcgtt gatgatggtg acatacagat gggattttgg 48900tgtggatgtc ctttctgttt gttagttttc tgtctaacag tcaggaccct cagctgcagg 48960tctgttggag tttgctggag gtccactccc tgtttgcctg ggtatcacaa gtggaggctg 49020cagaacagca aatattgcag aacagcaaat gttgctgtct gatccttctt ctggaagctt 49080tgtctcagag gggcacccgg ctgtatgggg tgtcagtcgg cccctactgg gaagtgtctc 49140ccagttaggc tactcggggg tcagggaccc acttgaggag gcagtctgtc cattctcaga 49200tctcaaactc tgtgctggga gaagcactac tctcttcaaa gctgtcagac agggacgttt 49260aactctgcag aagtttctgc tgccttttgt tcagctatgc cctaccccca gaggtggagt 49320ctacagaggc aggcaggctt ccttgagctg tggtgggctc cactcagttc gagtttcccg 49380gcggctttgt ttacctactc aagcctcagc aatggtggac gcccctcccc cagcctcgct 49440gctgccttgc agttcaatct cagactgctg tgcaagcagt gagcaaggct ccgtgggcgt 49500gggaccctct gagccaggcg agggatataa tctcctggtg tgccgtttgc taaggccatt 49560ggaaaagcac agtattaggg tgggagtgtc tcgattttcc aggtaccgtc tgtcacggct 49620tccctttgct aggaaaggga attcnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 49680nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 49740nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 49800nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 49860nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 49920nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 49980nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 50040nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 50100nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 50160nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 50220nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 50280nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 50340nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 50400nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 50460nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 50520nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 50580nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 50640nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 50700nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 50760nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 50820nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 50880nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 50940nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 51000nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 51060nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 51120nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 51180nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 51240nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 51300nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 51360nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 51420nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 51480nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 51540nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 51600nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 51660nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 51720nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 51780nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 51840nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 51900nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 51960nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 52020nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 52080nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 52140nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 52200nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 52260nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 52320nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 52380nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 52440nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 52500nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 52560nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 52620nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 52680nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 52740nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 52800nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 52860nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 52920nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 52980nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 53040nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 53100nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 53160nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 53220nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 53280nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 53340nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 53400nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 53460nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 53520nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 53580nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 53640nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 53700nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 53760nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 53820nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 53880nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 53940nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 54000nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 54060nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 54120nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 54180nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 54240nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 54300nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 54360nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 54420nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 54480nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 54540nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 54600nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 54660nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 54720nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 54780nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 54840nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 54900nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 54960nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 55020nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 55080nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 55140nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 55200nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 55260nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 55320nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 55380nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 55440nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 55500nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 55560nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 55620nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 55680nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 55740nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 55800nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 55860nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 55920nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 55980nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 56040nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 56100nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 56160nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 56220nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 56280nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 56340nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 56400nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 56460nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 56520nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn aacatcaact tctttctgtc 56580atgcatgtca cagattattt tcccccattg atcttttatt tttttatttt attttttttt 56640ttgagacaga gtctcactct gttgcccagg ctggagtgta gtggcacgat ctcggctcac 56700tgcaacctct gcctccaggg tttacaccat tctcctgcct cagcctcccg agtagctggg 56760aatacaggcg cccaccacca cgcccagcta atttttttgt atttttagga gagatggggt 56820ttcactgtgt tagccaggat ggtctcgatc tcctgacctc atgatccgcc ctccttggcc 56880tctcaaagtg ctgggattac aggcgtgagc caccgcgccc cgcctgatct tttaacttta 56940tggtggtgtt tgccatacag aattttttat tttttatttt tattcattta ttttttaatt 57000ttttttgaaa cagagtctca ctctgtagcc caggctggag tgcagtggcg cgatctcagt 57060tcactgtaag ctccgcctcc caggttcacg ccattctcct gcctcagcct cctgagtagc 57120tgggactata ggcgcccgcc accacgcccg gctaattttt tatatttttt agtagagacg 57180gggtttcacc atgttagcca ggatgtctcg atttcctgac cttgtgatcc acccacctcg 57240gcctcccaaa gtgctaggat tacaggcgtg agccaccgcg cctggcccag aattttttat 57300ttttaagtaa ctccattcac cacttcttaa aatgtaaagc ttattttcaa atgcatttcc 57360cactacccaa gactatataa ccccatctct actaaaaata caaaaattag ctgggcgtga 57420catgcctata gtcccagtta ctctgaaggc tgattcagga gaattgcttg aaccgagagg 57480cagaggttgc agtgagctga gatcgcgcct gccatgtcct ggatgacatc tgcctgggga 57540cactcgaggc agacacagcc ccggttgcag gctagccact ggtctgtgca tcccctcatc 57600ctaggcagat tgggcacagg gccaggcagg ctgtggggag ggctctccgc ctggactcag 57660taccatccag ccattccctg aaggtgactt tgccccccgg ggatcccagg ccatatcttc 57720acacattggg tgcacccagc atctggtggg tggggtctgg ggatgctgca aaacacattg 57780cagcagacag catggcccac cgcagagaat tctccaaccc gaaggtccat agtgcccagg 57840ctgagaagac ctggtgtgga aaaaacagga tcgcatgaca gtcaagttgc ccatggcctt 57900ttgcaggttt ccttccttta atcaaaggat attttaaacg ttaaactttt attttgaaat 57960aactttaaac ctttacagga aagttgcaaa aatgacattg ttatgcatta ttaacatttt 58020gtctggctta ctttattatt ccctctaaac tcatacagat gatgatgact tttgagcgtt 58080ttgaaactaa gttgcagaca ccgtaaccct ttatcactaa acatgtgaac aaggatattc 58140tcttgcgtac tcccagcaca gtgatcaaaa tcggggattt tacactgatg tcctggtgct 58200atctactccg cagcccacac tcagatgtca ccgattgtcc caataacatt cttgttttct 58260gatccataac ccaacccagg gccacacatt gcgctcatta ttgtatttgt cctcttgggt 58320ctggaacagg cctctgtcct tgtctttttg aggggtgcag cctagttact tgtagactgc 58380ctgcttgggt ttttctgctg tttgtccaca gtttgattga ggctgtgcct tttcctggag 58440tgaggctgtg ctcttctccc tgcgtcctgc ccggtgacgc agtctctgca tcccattcct 58500ggggatattc acttgataaa gggcgtgtct gtcaggactc ttcaggtgaa gttgggtttt 58560ttcccctatt aagtttcttg gccgcttgtg gtagctcacg cctctaatcc cagcactttg 58620ggaggcccag gtgggcggat cacctgaggt cagaagtgcg agaccagtct gggcaacata 58680gtgaaaccct atctctacta aaaatacaaa aattagctgg gtgtggtggc acactcatac 58740ctgtaatccc agctacttgg gaggctgaga cagaattgct tgagccgggg aggcggaggc 58800tgtagtgagc caagatcact ccactacact ccaggctggg caacagagca agactccatc 58860aaaaaaaaaa aaaaaagttt tggccaggcg cgatggctca cgcttgtaat cctagcactt 58920tgggagaccg aggtaggcag atcacaaggt caggagttcg agaccagcct gaccaatata 58980gtgaaacccc atctctacta aaaatacaaa aattagccag gtgtggtggt gtgcacctgt 59040aatcccagtt acttaggagg ctgaggcagg agaattgctt gaacctggga ggcggaggtt 59100gcagtgagcc gagatcgcgc cattgcactc cagcccaggc aacagagcga gactccgtct 59160caaaaaaaaa aaaagtttct tgtagggaga tacttggaga ttatgtgaat atcttgacag 59220aaacagttgt ttttatgaag attgtcgact tgggtttctc tcaccctgtt gtatcccagg 59280catttttgag ctcatctgag ctggaagccg ttctgttctg ggccctgagc caggtgaggg 59340tggttgaggc ccttgagttg ctggcaggtg ggtggggaag ctggtggaca ggacgacaaa 59400gtcagcaggc cctcacaggc tgcccagtgg gcatcatcag gggaggcaga ggcaaagtgg 59460cctgggcagg aggacccgat tggggagcac agaggcgaca ggtgggcagc attcagcaca 59520cgtgtctggg gttcagatgc cagatggaga catggcagca aggaggtgct gcagggtgcg 59580aggggaggca cagggagccg gtgagcagag ctgggtacac gtggagcgcg agggtctcag 59640cacctgattg gaacactggt gcgtttatat cgagagcgat ctgcagaagg actccccgag 59700ctgcttctag gcataaggac aagccctggc ccgataccca gtggggatgg ttgggcgaag 59760ctctgcctga tttctcagca gaggggtcga aggagccctg cttaggccca cgggtgggtg 59820agagagggga aaagccactc ttgacgggag gagacagtcc tgtccaggca tttattttct 59880ccaccgagga tgcgtataag agagccagct ctgggccaag ggccagcctg cctggggttc 59940cagttcttgc tccattggtc agtctctctg agcctcagtt tccctcgttg taaagtgtgg 60000tctgtgtcag ccatgtcttg aaggacagtc agttctgggc agccagttcc gggcagcttc 60060ctggggctgt ggtggccaga agccggtggg gctggcatcc acacacgtgt ccttggagtg 60120cccaatgctc cctctgtccc cacagaagca ggaggtatgc gggaacctga cgctgcagca 60180ccacatgctg gagcccgtgc agagggtccc ccggtacgag ctgctgctca aggactatct 60240gaagaggctc ccgcaggacg ccccagaccg gaaggatgcg gagagtgagc tggggccaag 60300ggctcccagg agggtgcagg ggcacctgcc aagaactctg ggggtgggga gagagggaga 60360gagagagtcg tggggtcatg ggggtagggg agagagaaac agagagacca gagggaaaga 60420gacagagaca gagacagagg caggggaggg agagagatgg agacagggag agagataggg 60480agatgtcagg gaaagagagg gagagggagg gagaaacaga ggggaggaga gagagacaca 60540cagagacaga ggaaggaggg ggagagagag agaaagagag agaacaagtg agccccctag 60600cagcccagga agggacttag catctttgag ggcagagaaa aatctcccct gcttgaagta 60660tgcgggtgga cattggtctg gggaaggtgc tactaaatgc tggccctgtt cagagcttag 60720gccccaggaa tgggcctcat agggctggtg ggggctgctc ctggtccaag ggcagcaact 60780ggtgtggctg gcaggcagcc cggacaggcg tctgtgcaga atcgtgggag ccccctgcct 60840ggtattggcc ccagaaggga tgtgtctgca gggtcagagg cagggtcacc cccttcctct 60900gggccagtcc tctagagtag cccaggcacc ctgggaaagg ctgagggcag agacagatcc 60960tctctctgga ggcagctgcc ctggagcccc tcaggcaagc aaaaacggag ccaccgtggc 61020cccaggctga acacagtctt cttcctgcag ggtccttgga gctcatctcc acagccgcca 61080accactccaa tgctgccatt cggaaagtgg tgagtgtggg gctccagtgg cgaccggcag 61140ggtggtgcag caagagtgag ggccaggggc ccttgtgggc cagccccttt gccgctatcc 61200tttggggggc tccacaagtg actcttttat acctccttcc acccccatcc ccaggggggt 61260cagctggaca gccccgcccc ttgctataat agacctgtgt ccttggtgag ttatttagca 61320ctctatgcct tagtttcccc atctataaaa tgggagcccc gacagagcct gtctcactgg 61380gcatctgtgg agagtaaaag aatcactcta tgggccgggc gcggttgctc actcctataa 61440tcccagcact ttgggaggcc gaggcgggcg gatcatgagg tcaggagttg gagaccagcc 61500tggccaatat ggtgaaaccc cgtctctact aaaaatacaa aaattagctg ggcgtagtgg 61560tgtatgcctg tagtcccagt tacttgggag gctgaggcag aagaatcgct tgaacctagg 61620aggcagaggt tgcagtgagc cgagatcgtg ccactgcact ccagcctgtg caacagagtg 61680agactccgtc tcaaaaaaaa aagaatcact ctaggccagg cgcggtggct cacgcctgta 61740atcccagcaa tttgggaggc cgaggcgggt ggatcatgag gtcaggagat ggagaccatc 61800ctggctaata tggtgaaacc ccatctctac taaaaataca aaaaattggg catggtggca 61860ggcgactgta gtcccagcta ctcaggaggc tgaggcagga gaatggcatg aacctgggag 61920gcggagcttg cagtgagccg agatcgcgcc actgcactcc agcctgggca acagagcaag 61980actctgtctc aaaaaaaaaa aaaaatgtat aacaaaaacc agggggaaaa aaaggaattt 62040cctttgtttt ctaagtatcc attatgttcc aggtactttc cctaggcagt ttcatttaat 62100tatactcttg accttgtgat ccgcccgcct cagctgggat tacaggcgtg agccaccatg 62160cccagcctat gatgggaatc cgcccgtcag caactttttg tattgccaaa cagtaagtgt 62220gaggctggct gtgccccaga ctcacagagc tctttaggcc cttttctgga gcatttagga 62280aagttatgaa aagcaatgtg tactcatagt gtgagtgcag ccacagaagc ttgagctgcc 62340cacagtggac acacaatcgc agggtcacag tggggacgtg acagcccaca cccatgcata 62400tgaggcactg cctgatcagc cccactgcct ggaatcccac agccagggca gacatccctg 62460acagtttcca tggtgatgcc ctggcacaga gcactgcctc ccacacacac agctcacata 62520aggtatgaac ttggctggcc tctgcctgat gaccctccat gtccagcccc gggccctggg 62580ccaggcttat ggagacatcc gtggtcacag tcctgccgca gaggcatcaa ggctcagtga 62640gagccacctg tcgtttgtga atgcgtctgt ccttggtgtg agcccatacg acatggagag 62700gcaccttcag tgtcttgggt aggcaaggaa gcagaagtgg gtggaggcca cgtgctgagg 62760gatgccctgg gtgggcagga cctgggcggg ttgtaggaac agcaggagcc caggcctggc 62820tgaggggatc cacacagggc gtctgagtgt gggacagggt cctggggagt ctaacccccg 62880ggttggtgcc cggacaggag aatgttctag ctggaagaac ctgcaaggcc attgtttaaa 62940atgttttaac ttgtgggaag acacagcttg gagatggcct tggagcaggg ctttgagcaa 63000atgccgcctc ttgagcctca cttggctctg ggcagagaga tcagagaagc tgtcaggcgc 63060tgctggccac acaggcaatg cccctattgg ccgcctgcct ccgtcacagg cgagaaggta 63120attggggctc agggcctcag tcagtgtccg ggcatctgct ggaccaggct gtggccagca 63180gacccgctgt gcctcgatgg gtgcgcccag ctgggtggac agcaccgcct gccccactcc 63240ctttacttcc cctggcccag ggttcagctg cctcaggttc tgcagcagcc tggggagcca 63300ggtactcagg gtcacaattc cagttctggt ctcttgtctc tgtcccttac ccacaccaag 63360ccagagctgg gcccacgaca gaccaccaaa caaccacaga cctcagctcc agcccagggc 63420ctccctacga acagtggatg tggcctggcc tggccctgtg ttcctcactg gtgtcttcct 63480gtgagcaaaa acagcagagt tgagactagg gtaggagctg ggacagctca gtgtctgtgt 63540caagctgcac agggtgaatg tggtgtgatg gaggtgagct cagtgtgcgt tttacaaagc 63600actcaaaagc aagttcattc tctcatcaac aacagtgatt tttgctacag tataccaaac 63660ttcaacttac ttcctgagcc caatttcttc agtggtatag tccctgtaag cccctttcct 63720aggagaagct acacccaagc caggaagttt tgcaaggggc cttgcagaat tcaaagcctt 63780ccctgatggg caagcgtgga gttgctctta acagaaacac aaaatgtcag aatccaaaag 63840gtctcttagg gcctcagacc caccatgggt gacacacgaa cccctccaag gagccagcga 63900ggcatctaag ggcacccttg acaggccagt gccagggcct cttctcaggc actgcccctc 63960ctgggacgag gccacgccct gtgtccaccc tcggggctcc agggcagggc tggagcagag 64020gcatcaggcc tcagcctggc caccctcctg ttggaatagc cccaggcagt gcctgcgcct 64080cacagcctgt gtcccctgct gggtcatttc aagcagctga tgaaactgac tcggtttcat 64140cagggcctct tactgggctc caattctgtg taactcagct gtgccccccg ttcctggatg 64200tgcagctcaa tttgctcaag aaaaaatgct ggagggggca gccccgttgg cccttggtgg 64260agaccacttt ctgggtcaac gctggtgtgt tttgggcgct ttgggggtga agtcactcgg 64320tcagatactg aggaagccac agttctccat gttcctggag agacttgggc ccagcttttc 64380tgatatcccg acatccacaa cgaaaacctg agctcccttc tttgggtcaa cctgcgtggt 64440aacttcattc acaaaggggc gtgtgcatgg ccgagagagc atccctcgct actcaggtgg 64500gcctcttgcc tctcagctac tgcgtcatgc ctacaaattc agccacacct tgatgatggg 64560agctggaggg ttggcctcga cgggctgcgg acctcagctc catgggagct ggtgcagggc 64620caggacccac cccaagtgct cagctcagtt ccatagctgg gaagatttgc cagataaagg 64680aagccagtgg gactcccggc actgcctcag ccctgagcca cactcactgc ttccagaatt 64740gtcctctgtc ccttcactgg gacccggtcc cactggtcct cagaggccaa gctcttgctg 64800tgcacccagc cccgcaggta cctgtcctcc cacatggttg ttctttgctc cttcagcccc 64860tgctgccccc tgccccatgg gcacgccctg tgttgttctc agctaccatg cagtgctgcc 64920cctgggtgcc gtggccaggg tcccccttcc ggggtcaaga gcaggcagtt ctgggctgag 64980gtgcacatcg gggctgccct tccactaatc actcccactt cccagcctct ctgggtgccc 65040acctggagaa gaggtttttg ccgccaggtc agtgatgccc attccccacc tagtgtgctt 65100ggagccccca ctgtggtctt gggtgtgaga gaaactgcag agggagccca ggccgcctca 65160cctgtgtctt tggagcaagg cccattcaac agaggtgccg ggggctgcag cacagcgagt 65220gagagtccct gtcaccctca agccagccct gcccttgggc tcctgtgcca agcaggacag 65280gtgctggggg gacctgcagg tgggccaatg aacggggacc acaaaccagg ccacacagct 65340ggaagtgccc ggccagaatt tgcacagagg cactggtgtc agtttgttgg ggctgccaga 65400acaaaatact acctgaggct gaagtctgag atcaaggtgt gggtagggtt ggtttatcct 65460gaggcctcac cttggcttgg agatgtcagt ttcctccttg tttccacaca tggtcttccc 65520tctgtgtctc tgtgtcctca tctcttctta taaagacacc agtaagattg aatcagggcc 65580caacctaaga cctcatttta acttaatgtc tctttaaagg ccctgtctcc aaatatagtc 65640acattcagag ttactgggaa ttatattttc aacatacgaa tttgggggga catagttcag 65700cccatcacag cctccaatgc ttatgcccaa tctgagccac ccctttcttg cttctttacc 65760cccaaccagt tcagttcatc tttttctttc tttctttttt ttttttttga gttgggatct 65820tgctgtgctc tccaggctgg agtgcagtgg tgtgatcaca actcactgta gccttgacct 65880cccaggctca agcgatcctc ctacctaagc cccccaaata tctgggacta caggcacgtg 65940ccaccacatc agctaatttt ttggattttt gttgagacgg ggtctcacta tgtcgcccag 66000attggtctcg aacttctggc ctcaagcaac cctcccgcct cagcctccca aagtgctgga 66060attacatgtg cgagccactg cgcctgggcc tccttctcct taaacctgaa gagtttatgt 66120taaacttttt ttaagcgtat gttgtttagc attttcctaa ggcagttttg gacccataca 66180agtggggata accgcaatga gtatccctcc ccagctccag aaacctcagc ctggtgctgt 66240tcttatggcc cctcagcccc tagccccgac acacacacac acacacacac acgcacacac 66300acacacacac acacacccca attttctgta atattttaaa gcatatccca gacctctttt 66360tattctgctc agctacagta atggtaagct tattttaggt agttttcttt taaacaggaa 66420aaaaaaaaaa agaaagaaaa acacctgcag gcttcagttt ccctgactgg agaggttgag 66480ataaacattt ctagtggcag ttccagctct gacatcctgg ggaactcgat acatatcttt 66540gccttttcct aaagtcttct taacagtggt ttcggcaccc ataacaaagt gtctcaaggg 66600aggcacctcc tccccagaga ggaaatgctg ggtgtggacc aagcttcaaa agcccaggaa 66660gttatttcat caaatgatgg ggcctttcca ggggttgcag gctattgtaa caggaaacgg 66720taattagcaa cttgccaaac gcagtcgggg ttttatgtac tgtgatgagg cttacgaccc 66780agggaccagt cacccatccc attttacagg tgaggaaact gaggctcaga aaggagaggt 66840gagtcatcca gggttgcagg gctgacagga ggcaggagac tggggtctct ccacctggca 66900ctgttcataa tcagacaggg aaaccaaggc tggactgggc agccccaggc tacacctgct 66960ccagcctcca caccaccctc ctctgtaggc agttcaggca caggagacag aggagggtac 67020agcccctgac cctggcctca gctcttcatg gcttcctcac atgtcctcac agtttttttt 67080tttttttttt tttttttttt tttttgagac agaatctcgc tctatagccc aggctggggt 67140gcaatggcgg gatctcagtt cactgcatca ctgcaaccaa cctccgcctc ctgggttgaa 67200gcaattctcc tgcctcagcc tcccgagtag ctgggattac aggcacccac taccactacc 67260atgcccggct attttttgta tattttttag tagaggtgga gtttcaccat gttggccagg 67320ctggtctcaa actcctgacc tcaagtgatc tgcccacctc agcctcccaa agtgctggga 67380ttacaggcat gagccactgc gtccagcctc cccctaaaat gttgccatgc ttgtctgcac 67440actcaggcat gtgggtgcac acctatattc atccatgtgg cgtgcctgga gggtgtggcc 67500cgcggtgccc agtatagagc aggcgcatgg ccacacggct ccagttgcag acagggatgc 67560ccccgtcccc agggctgcag gccatggctg tgctgcccac tgcccaccct agtaagtgct 67620gtgttcctcc agctgtggct cctttcaata gtttctcaga tgcaccagat aaagtcattg 67680gccaagttgt tccaaacatg aagatcttga tccacttaaa atctttactc ggtgccatgg 67740gcacaggcag ccgctgggag gtgagggagg gaagnnnnnn nnnnnnnnnn nnnnnnnnnn 67800nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 67860nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 67920nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 67980nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 68040nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 68100nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 68160nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 68220nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 68280nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 68340nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 68400nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 68460nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 68520nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 68580nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 68640nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 68700nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 68760nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 68820nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 68880nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 68940nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 69000nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 69060nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 69120nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 69180nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 69240nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 69300nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 69360nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 69420nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 69480nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 69540nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 69600nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 69660nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 69720nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 69780nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 69840nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 69900nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 69960nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 70020nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 70080nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 70140nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 70200nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 70260nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 70320nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 70380nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 70440nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 70500nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 70560nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 70620nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 70680nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 70740nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 70800nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 70860nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 70920nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 70980nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 71040nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 71100nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 71160nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 71220nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 71280nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 71340nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 71400nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 71460nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 71520nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 71580nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 71640nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 71700nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 71760nnnnnnnnnn nnnnnnnnnn ncccctcccc ttctcctcct ctctttctct tcctcttttc 71820ctcctccact cctctttttg agagtagctg ctgctttccc tgacttcctt cctcaagaat 71880acagtctatt ctggggggtt gcaggacagc tgaaaaagaa aacgtgagtt tccagaaaag 71940cacatcgccc tttctctcca ctcgacacag cctgcggttt gcagtgccag ctgcacgttg 72000gagtctcctg ggaagcttct agtgttgatc tccctcctag aggctcagtg aggtgatcgg 72060ggggtcagtg tgttttcaga gtcccagctg ctctgactta cagctgtggt tggagccctg 72120acctaggtcc ttggtctaag aagtcaagga tgtaagtcta ttaggactga gtgttgaatg 72180gtcttgatgg tgaagaagga ccttgcactg cctaccgagc tccctcatgc ctgcaattta 72240aataccgagg tgtgttctga gagccccagg tccttggggc acgggcctcc agggccctgt 72300aaggtggact ctggcctcga aggggggccc aggtctgagg ggtccagctc tggcaggggg 72360tgtctaagcc acgtccaaat ggtggacagt agctccaaca gtgacatgac agagggaggg 72420cctgtgtgtg tggtgtgtgt gtgtgtgctg tgtgtgcacg cacgtgcatg tatatggtgt 72480gtgtagaact gacacgcttg aagtccctgt cccgtgaccc accctcaggt acttgctgtt 72540gggttttcct atgattttga tgtgtcctga cccagcatat ggatgccctc cccttggagg 72600gcccagcctg caaaccccat ctgataccac acatgcctat gtcctgcagg cttctagtga 72660cccttgcaag ccgggtgagg ctggtcccag tctctgtgca gatcttggcc atgtctccac 72720agccagctcc ccccaagcag tggccctggc cctaccccag cctcctcaga acaggggtga 72780ccactgcagg ggagactgcc cctaacctgt gtctttgtgt ccccagaaga cacccactgc 72840agacccccag cccagcctgc tctgcggccc cctgcggctg tcagagagcg gtgagacctg 72900gagcgaggtg tgggccgcca tccccatgtc agatccccag gtgctgcacc tgcagggagg 72960cagccaggta cgtgtcccca ccccaccagg ccctcaggcc agcagcccag agcctcaggc 73020ctgaggcacc cacagcaggc ctgtgccacc agctggggtc cacctgcttg agcagtgtta 73080cctggggcct cagtttcccc agcagcgcag gattccctct gcttgccagt cacaggagac 73140agaacctact ggcacctggg tggaccccag agccagccag ggtgcaagga ggggtgggcc 73200aagggggcag aaagggtggc caggagccac agaacctgtc aggggagact caggaaccca 73260cacagccctg gcccggcccc ttggagcccc agttgctact ctgtcacagg ggctggcctg 73320tcctgcctcg cctccccagg ggccagacag cccaaagaag gccatggagt ctgggggtct 73380ggtgaggtgg gcaaggcatc cccctcccta tatctccctc gggaagaacc agtggatgcc 73440cccaccgacc gtcctcctgt tgacaaaagg cagtgcaggc acagccacca gacccctcgg 73500gctcagcacc tgccttgcgt tgcaagggtc tgggagtggc ctcctgggag aggcccccat 73560cactgaggtg agcccgaggc cgggaagctg tggccccgct gaccatctgc tcctctgctg 73620caggacggcc ggctgccccg caccatccct ctccccagct gcaaactgag tgtgccggac 73680cctgaggaga ggctggactc ggggcatgtg tggaagctgc agtgggccaa gcagtcctgg 73740tacctgagcg cctcctccgc agagctgcag cagcagtggc tggaaaccct aagcactgct 73800gcccatgggg acacggccca ggacagcccg ggggccctgc agcttcaggt ccctatgggc 73860gcagctgctc cgtgagctga gtctcccact gccctgcaca ccaccacatt ggacctgtgc 73920tgtcctggga ggtggtgttg gaggccccat gaagagcgcc ctggactgct gagggtgggc 73980caacagccca gagctcagga catttggctt tggggggaag gaaactgagg cccagagagg 74040ggcaaccact ggccaagggt cacccagcaa gttttggcta agagcctggc ctccagcccc 74100agcagtgtgg cccagagcag gggccgactg ccaaagtaac catcatccat atgggccgtg 74160tggtgatgct ggcccggaag gcagaaagag gcagcatggg cactgccagg gacagccaca 74220tcctgctggt ctgcagcgtg gtccaccccg cctctgccca gcctgtctac accgtgtgag 74280ctgaatcgtg acttgcttcc cacctccttt ctctgtcctc tcctgaggtt ctgcctgcag 74340cccccaggag gtgggcctgc cccatcctag ctggactcat ggttcctaaa taaccacgct 74400cagaagctct gctaggactt accccagcca ctgagtggca ggcgcatgag atttgtggct 74460gttcctgatg ctagtggcac acagtgctta tctgcataaa taaacactgg ccaccagcag 74520tgggcgcagc ctcggtgatc tctccccagc ccctcagcag cgtcccccca caaggtgggc 74580tggctgataa agggttgagg gacaagggtg atggaatcac atcggccaca caccacctgg 74640aaagcagttc tgcatctgtt gtcctgccag ggggtgggca ggtggcaggt gaagatggcc 74700aggctcagag atggggaggg tggggtgcag ggcactcaag atggtgccag tggggacctt 74760gcaaaggcta cttggggtgg gggtgctgtt tgcatcagag ccctgagggc ctctgggaga 74820gagtggggga atttagggac cagtgattgg gggtgctcac tggtcaggac ccccacccct 74880ctaatatcca tatagcacca tgtggagaca cctgggaaga caagcagggc acctacctgg 74940gggactgaga gggcaggagg aagagccaga aatgggagac tggtcacagg tgatgggggg 75000gacaggtgag atgagaacac aggtgaggcg gagacagctg agatgggggc acaggtgagg 75060gcacaggtga gctcccacga gctctagctg ggccacgctt gggatgctgc tgcagccact 75120gcacacggag cccctgctgg ctacctgggc ctctgccaca gcttcccatg ctatgctgga 75180tgttctggtc ccacttcaca ctaaggaaac tgaggctcag ggatgctaaa caacctgcat 75240acttccacat tggacagtgc ccaccagggg gagatggaat cagacccttt cagctgcaca 75300gcagcgtctc aatcggctcc agctgctgcc caaggaagca gcaaagcccc tcacccagct 75360caggctggag cagggacggg gtggaggagg cctcgagggg accccagccc gttggccctg 75420ccttctcccc tgcagagctg agctctgacc cacattcatc ccctacagag cagtgccgga 75480gccgctccct cacacacgtc tactaagcat cggctgcagc cctaatgagg cccttctttg 75540aagggtcagg actccgtcct ctgcctgctt cagtagttct atttctgcca tctcaccaca 75600accccaggaa atccccactt gacagctgag gaaactgagt catagagagg ccacccccag 75660agccccacag ctccggaacc gagcaggcac ctggaattgg cctgccggcc atggtgaatc 75720cagcagggca ggtgtctgct ggggccaaag actgtgctgt ggggtcgcca tggagagtcc 75780cctagcctcg agccctctct cttgcaccca ccaacctggc tagtccgctg ggaggcagag 75840acaaggtctg tcaccgcagg cctgggggca gggcgtgccc ccagactctg ccgcctgctg 75900cgaggccagt gctgatggga gcttgtttgc tgtcatactg gtggctgtag tctgttccca 75960ctttttccaa aagaggaagc tgaggctcag acaggggcag tgagaggcct ccagcatgca 76020gagcctgcgt gctgtgccct tacagagctg gccttcctag ggtctgttcc cagagacccc 76080tcctgcggga caccccagca aggatgctgc tggggagctg gctgcctggg cctggcctct 76140cgccacacac tgccccgccc ccagctccag ccctcccatc ccacagccca gggcctgggc 76200acactccact gcccgtccca tggagtggca gagcctggac acatgctggc tggtcgcatg 76260ctgnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 76320nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 76380nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 76440nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 76500nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 76560nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 76620nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 76680nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 76740nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 76800nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 76860nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 76920nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 76980nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 77040nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 77100nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 77160nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 77220nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 77280nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 77340nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 77400nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 77460nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 77520nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 77580nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 77640nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 77700nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 77760nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 77820nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 77880nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 77940nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 78000nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 78060nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 78120nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 78180nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 78240nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 78300nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 78360nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 78420nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 78480nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 78540nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 78600nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 78660nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 78720nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 78780nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 78840nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 78900nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 78960nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 79020nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 79080nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 79140nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 79200nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 79260nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 79320nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 79380nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 79440nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 79500nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 79560nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 79620nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 79680nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 79740nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 79800nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 79860nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 79920nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 79980nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 80040nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 80100nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 80160nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 80220nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 80280nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 80340nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 80400nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 80460nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 80520nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 80580nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 80640nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 80700nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 80760nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 80820nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 80880nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 80940nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 81000nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 81060nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 81120nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 81180nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 81240nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 81300nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 81360nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 81420nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 81480nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 81540nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 81600nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 81660nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 81720nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 81780nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 81840nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 81900nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 81960nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 82020nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 82080nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 82140nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 82200nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 82260nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 82320nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 82380nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 82440nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 82500nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 82560nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 82620nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 82680nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 82740nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 82800nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 82860nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 82920nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 82980nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 83040nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 83100nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 83160nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 83220nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 83280nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn ctgggcatgg tggcgggcgt ctgtagtctc 83340agctactcgg gaggctgagg caggcagaat ggtgtgaacc caggcaggcg gagcttgcag 83400tgagccgaga tcgtgccact gcactccagc ctgggcgaca gtagtgagac tccatctcaa 83460aaaataataa taataaatta aaaaaaaaaa aagactagct tgaccaatat ggtgaaaccc 83520tgtctctact aaaaatacaa aaattagttg ggtgtggtgg cacacgcctg tagtcccagc 83580tactggggag gctgaggcag gagaatcact taaacccagg agatggaggt tgcagtgagc 83640tgaggtcgca ccactgcact ctagcctggg cggcagaacg agacttcatc tcaaaaaaag 83700aaaaaaaaaa aaagaacaga atgttggtag aaataggaac atcacaggtg cttctggtga 83760ggccttagaa gagagtaagg aacatgctat tggaaagagg aggaaaagtg atctttgtta 83820tagagtggta gagaagttgg ctgaactaca ttctagtgtt gggtagaaag taggagactt 83880ccagctgggc gtgttggctc acgcctgtaa tcctggcact ttgggaggcc gaggcgggca 83940gatcacgagg tcaggagatc gagaccatcc tggctaacac ggtgaaaccc catctctact 84000aaaaatacaa aaaaaaatta gccgggcatg gtggcaggcg cctgtagtct cagctactca 84060ggaggctgag ggaggagaat ggcatgaacc caggaggcgg agcttgcagt gagccgagat 84120cgcaccactg cactccagcc tgggtgacag aacaagactc cgtctcaaaa aaaaaaaaaa 84180aaaagaaagt agatttccaa acagtgtaga aggtatggcc tggtttgtcc tttgctgctt 84240gtagcaattt atctctttta agagaaaaga gataaattga ggaaggaact gttaagcaaa 84300aaggaactgt acttggtaat ctaggaagcc cttagcctgt ccagattgct aaattagcaa 84360attcactatt gagaaaatgt ggagagaaag tgtgaagaga acaccaaggg tgttgttgga 84420cattttgttg aagacattat gaatgtgact cgtggatcca atcaaccatc tcagtggaat 84480tgaggaatag agatgaggtt atccaggaat gatctgtgga tagagcctct tgtctgatgg 84540catgggtccc tgtaaactac acaggagacc aacaaggttt ctgagaatgt tacaccagca 84600gaaatgctgc cagcctggat tgaaaggcat gggggtgcta ggcatggtgg ctcatgcctg 84660caatctcagc actttgggag gccgaggtgg gtggatcacg aggtcaggag atggagacca 84720tcctggataa cacggtgaaa accccgtcta tactaaaaaa aaaatacaaa aaaaattagc 84780caggtgtggt ggcaggcgcc tgtagtccca gttacttggg aggctgaggc aggagaatgg 84840cgtgaacctg ggaggcagag cttgcagtga gctgagaccg caccannnnn nnnnnnnnnn 84900nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 84960nnnnnnnnnn tgctgggcct cagacttgct taggaccaag tgatcctttc gtttcttccc 85020ttttctccct tttgtttttt gttgtggttg tttgtttgtt ttttgagatg gagtctcgtt 85080ctgttgccca ggctggagtg cagtggtgcg atctccgctc actgcaaact ctggctccca 85140ggttcatgcc attctcttgc ctcagcctcc tgagtagctg ggactacagg cacccgccac 85200catgcctggc taattttttg tatttttagt aaagatagag tttcgttagc cagggtggtc 85260tcaatctcct gaccttgtga tccacccgcc tcagcctccc aaagtgctgg gattacaggc 85320atgagccacc gtgcctggcc ccttttctcc cttttggaag gcgctcagac tttggaggat 85380attgggatga ggtgaatgtg ttttgaacat gggaccacag taacctttgg gagttccaac 85440tgtagtgggt tcaatagtgt acctgcaaat ttatgtctgc ctggaacgtc agaatgtgat 85500cttatttgga aataagatct ttgcagatat agttagatat agaagtcata ctggattagg 85560atgggccata aatccaataa ctagtgtccc aataagaaga aaagacacag agatgtggtg 85620gtggaggtag agattggagt ggtgcagcta caagccaagg agtaccaaga atgcctttgt 85680tttgaggccc cgatttgtgg tactttgttg cagtgaccgt gggacactaa cacacagggt 85740gggtaattaa gctattaggt ctggaggcaa ctggaactta gtctgctggg gagtctggta 85800gctactactc catcctccag ttaagagccc cagaacacat ctgctgatga acaaagttgg 85860atttgcagac ttgctgcaat gagcgggagc acatgtgttg ggaaagccta gggcattgca 85920atgggatgct tgtagggagg acgtagagta ggatttgggc tgtgttaggt ggttttgaag 85980ggggttcaaa agccagtctg cactggatgc tatcgggagc aattctatgc ttgggtattt 86040taatacattc catcagaaag cagaggaatg aaatgaggct caagctgcaa ttggtatgaa 86100acagcagcca ctcgtatcac ccaggatatt ggtattgtct ttccagggac ttttttcctt 86160tttttttttt tttttttttt tttgagactg agtctttctc tgtcacccag gtggagtgca 86220atggcatgat ctcggctcac tgcaacctct gcctcccggg ttcaaacgat tctcctgcct 86280cagcctctca cgtagctggg attacaggtg cctactacca tgcccagcta atttttgtat 86340ttttttagta gagattgagt ttcgccatgt tagccaggct ggtcttgaac tcctgggctc 86400aaggttatcc atctgcgtca gcctcccaaa gtggtgggat tacaggcgtg agcctccaca 86460cttggcctgt gactttgctt tttctgtgcc aagacaagat gttgaagtgg tcttgtctga 86520tattgatgcc ctgtgagact gtttatgttc aacgggagct gtgaatgcca gctcagttct 86580ggcttctcac agggtagaca gtcaccccgg taccatccag ctgggcattt accctccaac 86640acctgggaga caccaggaga cacttggaga cactggttga gggctgctct tgggaccact 86700aattcattcc cctacatttc tggcctgccc tgcatgagca gaggggcttt ggacttgttt 86760tgttttgttt ttttttgttg ttgttgtttt tgagatggag tctcactgtc ccccaggctg 86820gagtgcaatg gcgtgatctt ggctcactgc aagctctgcc tcccgggttc acgccattct 86880cctgcctcag cctcccaagt agctgggact acaggcaccc gccaccatgt ccggctaatt 86940ttttgtattt ttagtagaga tggggtttca ctgtgttagc caggatggtc tcgatctcct 87000gacctcgtga tccgcccacc tcggcctccc aaagtgctgg gatcacaggc gtgagccacc 87060gcgcctggcc tggacctgtc tgacaatgat tccggcttga ttattattca ggtatggtga 87120ggccagcaga tcagtagaca actaccattg agaagatagc ttgctatcac agatcccaag 87180tggaagaggc ctccaccctt gaagggccac acagggaagc accagggttg gtcaggaggc 87240agagggaatg ggggaagtgt gggccggagc ctttactgtg gttttgaagg aaggcatggg 87300caagcagggc aagcaggctg agtactggct ggtgtgaatc atttcagtgg gctctggggc 87360acagaggctc ttgctgagtg tctggtacct gcctcagggt gaggagtgca gggggatggt 87420ggcccagagt gtggtagccc tggagaggag gtgtaggatg tgggctctgg gttggttggt 87480ttgcacttga aaggcatgat cacaggcaag tcctttactc tctagggact ggctatccct 87540gggaggggct gtcactccag gtcagcaagt ccccaagatg tcaaagcatc agaatacaga 87600aaataaaaaa atatgattaa tatgggcccc cagagggagt ctccagccgc tagaagttgg 87660gctgaggccc ccgagtgggc atggctgagg agacatggca ggcacagacg gcatctgcta 87720caggtgacca gaccaaagtc agctggctca ggaccagggt ttcagctcag ggcagcctga 87780tgccaaattc cacattctga actatagctc ggtttggtga gaggagagaa aagaacaaca 87840ggtgttttcc agcgtcattg accacattac ctattcggac tgtgtctcac ttgcatgttc 87900tgcagaccat caccagtgcc tgctctgtgc ctggccccat gctgggctct ggggccacag 87960agatgagcca ggcatggttc ctgcccttga ggtatgattt gtctgcaggg gatttgagga 88020gggagcaaac ctgtggtcca gcccaaccct ggatggctag agaatccttg ctgaagaagg 88080tggtgcccga gctgagtgct gaagcaccag cagggccaca gagctgagca gagcgggcag 88140gaaagtcact gcagctggaa cacagtgggg acagggctgg ggcagggaag t 88191 4 625 PRTHuman 4 Met Glu Ser Gly Arg Gly Ser Ser Thr Pro Pro Gly Pro Ile Ala Ala1 5 10 15 Leu Gly Met Pro Asp Thr Gly Pro Gly Ser Ser Ser Leu Gly LysLeu 20 25 30 Gln Ala Leu Pro Val Gly Pro Arg Ala His Cys Gly Asp Pro ValSer 35 40 45 Leu Ala Ala Ala Gly Asp Gly Ser Pro Asp Ile Gly Pro Thr GlyGlu 50 55 60 Leu Ser Gly Ser Leu Lys Ile Pro Asn Arg Asp Ser Gly Ile AspSer 65 70 75 80 Pro Ser Ser Ser Val Ala Gly Glu Asn Phe Pro Cys Glu GluGly Leu 85 90 95 Glu Ala Gly Pro Ser Pro Thr Val Leu Gly Ala His Ala GluMet Ala 100 105 110 Leu Asp Ser Gln Val Pro Lys Val Thr Pro Gln Glu GluAla Asp Ser 115 120 125 Asp Val Gly Glu Glu Pro Asp Ser Glu Asn Thr ProGln Lys Ala Asp 130 135 140 Lys Asp Ala Gly Leu Ala Gln His Ser Gly ProGln Lys Leu Leu His 145 150 155 160 Ile Ala Gln Glu Leu Leu His Thr GluGlu Thr Tyr Val Lys Arg Leu 165 170 175 His Leu Leu Asp Gln Val Phe CysThr Arg Leu Thr Asp Ala Gly Ile 180 185 190 Pro Pro Glu Val Ile Met GlyIle Phe Ser Asn Ile Ser Ser Ile His 195 200 205 Arg Phe His Gly Gln PheLeu Leu Pro Glu Leu Lys Thr Arg Ile Thr 210 215 220 Glu Glu Trp Asp ThrAsn Pro Arg Leu Gly Asp Ile Leu Gln Lys Leu 225 230 235 240 Ala Pro PheLeu Lys Met Tyr Gly Glu Tyr Val Lys Asn Phe Asp Arg 245 250 255 Ala ValGly Leu Val Ser Thr Trp Thr Gln Arg Ser Pro Leu Phe Lys 260 265 270 AspVal Ile His Ser Ile Gln Lys Gln Glu Val Cys Gly Asn Leu Thr 275 280 285Leu Gln His His Met Leu Glu Pro Val Gln Arg Val Pro Arg Tyr Glu 290 295300 Leu Leu Leu Lys Asp Tyr Leu Lys Arg Leu Pro Gln Asp Ala Pro Asp 305310 315 320 Arg Lys Asp Ala Glu Arg Ser Leu Glu Leu Ile Ser Thr Ala AlaAsn 325 330 335 His Ser Asn Ala Ala Ile Arg Lys Val Glu Lys Met His LysLeu Leu 340 345 350 Glu Val Tyr Glu Gln Leu Gly Gly Glu Glu Asp Ile ValAsn Pro Ala 355 360 365 Asn Glu Leu Ile Lys Glu Gly Gln Ile Gln Lys LeuSer Ala Lys Asn 370 375 380 Gly Thr Pro Gln Asp Arg His Leu Phe Leu PheAsn Ser Met Ile Leu 385 390 395 400 Tyr Cys Val Pro Lys Leu Arg Leu MetGly Gln Lys Phe Ser Val Arg 405 410 415 Glu Lys Met Asp Ile Ser Gly LeuGln Val Gln Asp Ile Val Lys Pro 420 425 430 Asn Thr Ala His Thr Phe IleIle Thr Gly Arg Lys Arg Ser Leu Glu 435 440 445 Leu Gln Thr Arg Thr GluGlu Glu Lys Lys Glu Trp Ile Gln Ile Ile 450 455 460 Gln Ala Thr Ile GluLys His Lys Gln Asn Ser Glu Thr Phe Lys Ala 465 470 475 480 Phe Gly GlyAla Phe Ser Gln Asp Glu Asp Pro Ser Leu Ser Pro Asp 485 490 495 Met ProIle Thr Ser Thr Ser Pro Val Glu Pro Val Val Thr Thr Glu 500 505 510 GlySer Ser Gly Ala Ala Gly Leu Glu Pro Arg Lys Leu Ser Ser Lys 515 520 525Thr Arg Arg Asp Lys Glu Lys Gln Ser Cys Lys Ser Cys Gly Glu Thr 530 535540 Phe Asn Ser Ile Thr Lys Arg Arg His His Cys Lys Leu Cys Gly Ala 545550 555 560 Val Ile Cys Gly Lys Cys Ser Glu Phe Lys Ala Glu Asn Ser ArgGln 565 570 575 Ser Arg Val Cys Arg Asp Cys Phe Leu Thr Gln Pro Val AlaPro Glu 580 585 590 Ser Thr Glu Val Gly Ala Pro Ser Ser Cys Ser Pro ProGly Gly Ala 595 600 605 Ala Glu Pro Pro Asp Thr Cys Ser Cys Ala Pro AlaAla Leu Ala Ala 610 615 620 Ser 625 5 733 PRT Mus musculus 5 Met Glu LeuGly Arg Ser Ser Ser Thr Pro Gln Glu Glu Ala Ile Ser 1 5 10 15 Pro LeuGly Val Leu Gly Thr Gly Pro Ser Ser Ser Pro Leu Gly Lys 20 25 30 Leu GlnAla Leu Pro Ile Gly Pro Gly Ala His Arg Gly Ala His Ser 35 40 45 Ser SerAla Pro Ala Gly Asp Ser Ser Thr Arg Glu Pro Ser Gly Ala 50 55 60 Met LysIle Pro Asn Arg Asp Ser Gly Ile Asp Ser Pro Ser Ser Ser 65 70 75 80 ValAla Ser Glu Asn Phe Pro Cys Glu Glu Ser Ser Glu Gly Ser Pro 85 90 95 SerPro Ala Ile Leu Gly Leu Pro Ser Glu Thr Ala Ser Asp Ser Arg 100 105 110Val Pro Gln Asp Asn Pro Gln Glu Glu Glu Asp Ser Gly Val Gly Glu 115 120125 Glu Pro Asp Pro Lys Val Thr Leu Phe Arg Pro Gln Glu Asp Val Ser 130135 140 Leu Thr Gln Cys Ser Asp Pro Gln Lys Leu Leu His Ile Ala Gln Glu145 150 155 160 Leu Leu His Thr Glu Glu Ala Tyr Val Lys Arg Leu His LeuLeu Asp 165 170 175 Gln Val Phe Cys Thr Lys Leu Thr Glu Ala Gly Ile ProLeu Glu Val 180 185 190 Thr Thr Gly Ile Phe Ser Asn Ile Ser Ser Ile TyrArg Phe His Gly 195 200 205 Gln Phe Leu Leu Pro Glu Leu Gln Lys Arg IleThr Glu Glu Trp Asp 210 215 220 Thr Asn Pro Arg Leu Gly Asp Ile Leu GlnLys Leu Ala Pro Phe Leu 225 230 235 240 Lys Met Tyr Gly Glu Tyr Val LysAsn Phe Asp Arg Ala Met Gly Leu 245 250 255 Val Ser Thr Trp Thr Gln ArgSer Pro Gln Phe Lys Asp Val Ile His 260 265 270 Thr Ile Gln Lys Gln GluVal Cys Gly Asn Leu Thr Leu Gln His His 275 280 285 Met Leu Glu Pro ValGln Arg Val Pro Arg Tyr Glu Leu Leu Leu Lys 290 295 300 Asp Tyr Leu LysArg Leu Pro Arg Asp Ala Pro Asp Arg Lys Asp Ala 305 310 315 320 Glu ArgSer Leu Glu Leu Ile Ser Thr Ala Ala Asp His Ser Asn Ala 325 330 335 AlaIle Arg Lys Met Glu Lys Met His Lys Leu Leu Glu Val Tyr Glu 340 345 350Gln Leu Gly Gly Glu Glu Asp Ile Val Asn Pro Ala Asn Glu Leu Ile 355 360365 Lys Glu Gly Ser Ile Gln Lys Leu Ser Ala Lys Asn Gly Thr Thr Gln 370375 380 Asp Arg His Leu Phe Leu Phe Asn Asn Val Met Leu Tyr Cys Val Pro385 390 395 400 Lys Leu Arg Leu Met Gly Gln Lys Leu Ser Val Arg Glu LysMet Asp 405 410 415 Ile Ser Asp Leu Gln Val Gln Asp Ile Val Lys Pro AsnAla Ala Cys 420 425 430 Thr Phe Ile Ile Thr Gly Arg Lys Arg Ser Leu GluLeu Gln Thr Arg 435 440 445 Thr Glu Glu Glu Lys Lys Glu Trp Ile Gln ValIle Gln Ala Thr Val 450 455 460 Glu Lys His Lys Gln Lys Ser Glu Thr PheArg Ala Phe Gly Gly Ala 465 470 475 480 Cys Ser Gln Asp Glu Glu Pro ThrLeu Ser Pro Asp Gln Pro Val Met 485 490 495 Ser Thr Ser Ser Val Glu ProAla Gly Val Ala Asp Ser Asn Gly Gly 500 505 510 Thr Pro Gly Ile Glu SerArg Lys Ser Ser Ser Lys Thr Arg Arg Asp 515 520 525 Lys Glu Lys Pro GlyCys Lys Ser Cys Gly Glu Thr Phe Asn Ser Ile 530 535 540 Thr Lys Arg ArgTyr Arg Cys Lys Leu Cys Gly Glu Val Ile Cys Arg 545 550 555 560 Lys CysSer Glu Phe Lys Ala Glu Asn Ser Lys Gln Ser Arg Val Cys 565 570 575 ArgGlu Cys Phe Leu Glu Glu Pro Leu Val Pro Pro Ser Pro Ser Ser 580 585 590Glu Thr Pro Thr Glu Leu Lys Gln Asn Ala Glu Lys Pro Pro Ser Val 595 600605 Asp Pro Arg Pro Ser Leu Leu Cys Gly Thr Leu Asn Leu Ser Asp Asp 610615 620 Gly Thr Thr Trp Asn Glu Val Trp Ala Ala Ile Pro Glu Ser Asp Pro625 630 635 640 Gln Val Leu Asp Leu Leu Ala Gly Ser Gln Ala Gly Arg LeuLeu Tyr 645 650 655 Ser Ile Pro Leu Ser Gly Cys Asn Ile Thr Met Pro AspPro Glu Glu 660 665 670 Gly Leu Glu Ala Gly Cys Ala Trp Lys Leu His GlnGly Ser Gln Thr 675 680 685 Trp Trp Leu Ser Ala Pro Ser Thr Lys Leu GlnGln Cys Trp Leu Lys 690 695 700 Ala Leu Gly Thr Ala Val His Gly Asp ThrAla Gly Asp Arg Pro Gly 705 710 715 720 Ala Ser Gln Pro Gln Ala Pro AlaGly Thr Asp Thr Pro 725 730

That which is claimed is:
 1. An isolated polypeptide consisting of anamino acid sequence selected from the group consisting of: (a) an aminoacid sequence shown in SEQ ID NO:2; (b) an amino acid sequence of anallelic variant of an amino acid sequence shown in SEQ ID NO:2, whereinsaid allelic variant is encoded by a nucleic acid molecule thathybridizes under stringent conditions to the opposite strand of anucleic acid molecule shown in SEQ ID NOS:1 or 3; (c) an amino acidsequence of an ortholog of an amino acid sequence shown in SEQ ID NO:2,wherein said ortholog is encoded by a nucleic acid molecule thathybridizes under stringent conditions to the opposite strand of anucleic acid molecule shown in SEQ ID NOS:1 or 3; and (d) a fragment ofan amino acid sequence shown in SEQ ID NO:2, wherein said fragmentcomprises at least 10 contiguous amino acids.
 2. An isolated polypeptidecomprising an amino acid sequence selected from the group consisting of:(a) an amino acid sequence shown in SEQ ID NO:2; (b) an amino acidsequence of an allelic variant of an amino acid sequence shown in SEQ IDNO:2, wherein said allelic variant is encoded by a nucleic acid moleculethat hybridizes under stringent conditions to the opposite strand of anucleic acid molecule shown in SEQ ID NOS:1 or 3; (c) an amino acidsequence of an ortholog of an amino acid sequence shown in SEQ ID NO:2,wherein said ortholog is encoded by a nucleic acid molecule thathybridizes under stringent conditions to the opposite strand of anucleic acid molecule shown in SEQ ID NOS:1 or 3; and (d) a fragment ofan amino acid sequence shown in SEQ ID NO:2, wherein said fragmentcomprises at least 10 contiguous amino acids.
 3. An isolated antibodythat selectively binds to a polypeptide of claim
 2. 4. An isolatednucleic acid molecule consisting of a nucleotide sequence selected fromthe group consisting of: (a) a nucleotide sequence that encodes an aminoacid sequence shown in SEQ ID NO:2; (b) a nucleotide sequence thatencodes of an allelic variant of an amino acid sequence shown in SEQ IDNO:2, wherein said nucleotide sequence hybridizes under stringentconditions to the opposite strand of a nucleic acid molecule shown inSEQ ID NOS: 1 or 3; (c) a nucleotide sequence that encodes an orthologof an amino acid sequence shown in SEQ ID NO:2, wherein said nucleotidesequence hybridizes under stringent conditions to the opposite strand ofa nucleic acid molecule shown in SEQ ID NOS:1 or3; (d) a nucleotidesequence that encodes a fragment of an amino acid sequence shown in SEQID NO:2, wherein said fragment comprises at least 10 contiguous aminoacids; and (e) a nucleotide sequence that is the complement of anucleotide sequence of (a)-(d).
 5. An isolated nucleic acid moleculecomprising a nucleotide sequence selected from the group consisting of:(a) a nucleotide sequence that encodes an amino acid sequence shown inSEQ ID NO:2; (b) a nucleotide sequence that encodes of an allelicvariant of an amino acid sequence shown in SEQ ID NO:2, wherein saidnucleotide sequence hybridizes under stringent conditions to theopposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3;(c) a nucleotide sequence that encodes an ortholog of an amino acidsequence shown in SEQ ID NO:2, wherein said nucleotide sequencehybridizes under stringent conditions to the opposite strand of anucleic acid molecule shown in SEQ ID NOS:1 or3; (d) a nucleotidesequence that encodes a fragment of an amino acid sequence shown in SEQID NO:2, wherein said fragment comprises at least 10 contiguous aminoacids; and (e) a nucleotide sequence that is the complement of anucleotide sequence of (a)-(d).
 6. A gene chip comprising a nucleic acidmolecule of claim
 5. 7. A transgenic non-human animal comprising anucleic acid molecule of claim
 5. 8. A nucleic acid vector comprising anucleic acid molecule of claim
 5. 9. A host cell containing the vectorof claim
 8. 10. A method for producing any of the polypeptides of claim1 comprising introducing a nucleotide sequence encoding any of the aminoacid sequences in (a)-(d) into a host cell, and culturing the host cellunder conditions in which the polypeptides are expressed from thenucleotide sequence.
 11. A method for producing any of the polypeptidesof claim 2 comprising introducing a nucleotide sequence encoding any ofthe amino acid sequences in (a)-(d) into a host cell, and culturing thehost cell under conditions in which the polypeptides are expressed fromthe nucleotide sequence.
 12. A method for detecting the presence of anyof the polypeptides of claim 2 in a sample, said method comprisingcontacting said sample with a detection agent that specifically allowsdetection of the presence of the polypeptide in the sample and thendetecting the presence of the polypeptide.
 13. A method for detectingthe presence of a nucleic acid molecule of claim 5 in a sample, saidmethod comprising contacting the sample with an oligonucleotide thathybridizes to said nucleic acid molecule under stringent conditions anddetermining whether the oligonucleotide binds to said nucleic acidmolecule in the sample.
 14. A method for identifying a modulator of apolypeptide of claim 2, said method comprising contacting saidpolypeptide with an agent and determining if said agent has modulatedthe function or activity of said polypeptide.
 15. The method of claim14, wherein said agent is administered to a host cell comprising anexpression vector that expresses said polypeptide.
 16. A method foridentifying an agent that binds to any of the polypeptides of claim 2,said method comprising contacting the polypeptide with an agent andassaying the contacted mixture to determine whether a complex is formedwith the agent bound to the polypeptide.
 17. A pharmaceuticalcomposition comprising an agent identified by the method of claim 16 anda pharmaceutically acceptable carrier therefor.
 18. A method fortreating a disease or condition mediated by a human Ras-like protein,said method comprising administering to a patient a pharmaceuticallyeffective amount of an agent identified by the method of claim
 16. 19. Amethod for identifying a modulator of the expression of a polypeptide ofclaim 2, said method comprising contacting a cell expressing saidpolypeptide with an agent, and determining if said agent has modulatedthe expression of said polypeptide.
 20. An isolated human Ras-likeprotein polypeptide having an amino acid sequence that shares at least70% homology with an amino acid sequence shown in SEQ ID NO:2.
 21. Apolypeptide according to claim 20 that shares at least 90 percenthomology with an amino acid sequence shown in SEQ ID NO:2.
 22. Anisolated nucleic acid molecule encoding a human Ras-like proteinpolypeptide, said nucleic acid molecule sharing at least 80 percenthomology with a nucleic acid molecule shown in SEQ ID NOS:1 or
 3. 23. Anucleic acid molecule according to claim 22 that shares at least 90percent homology with a nucleic acid molecule shown in SEQ ID NOS: 1 or3.